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Graduate Theses and Dissertations
Graduate School
May 2014
Immature Myeloid Cells Promote Tumor
Formation Via Non-Suppressive Mechanism
Myrna Lillian Ortiz
University of South Florida, [email protected]
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Immature Myeloid Cells Promote Tumor Formation
Via Non-Suppressive Mechanism
by
Myrna Lillian Ortiz-Ruiz
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
Department of Molecular Medicine
College of Medicine
University of South Florida
Major Professor: Dmitry I. Gabrilovich, M.D., Ph.D.
Julie Djeu, Ph.D.
Tomar Ghansah, Ph.D.
Thomas Klein, Ph.D.
Peter Medveczky, M.D.
Date of Approval:
February 17, 2014
Keywords: MDSC, S100A9, T helper cells, inflammation, CCL4
Copyright © 2014, Myrna L. Ortiz-Ruiz
DEDICATION
I dedicate this project to:
To myself. I took this journey that included tremendous challenges that
eventually led to extraordinary learning experiences. I dedicate this work to myself
simply because I was able to reach my most desirable and amazing professional goal to
date, becoming a Ph.D. Becoming a scientist is an achievement I am so proud of and
know deserves recognition. Throughout these years, not only have I gained an
immense amount of scientific knowledge by accepting experimental and creative
challenges but, more importantly, I discovered much more about myself than ever
before. It brought all kinds of emotions, which gave me so much strength to overcome,
such that I believe I really can do what I put my mind and effort into. No matter how long
it took to get to this rewarding moment, I am here, and that’s what counts.
To my family. You were an unconditional supply of support, compassion and
motivation. Mami, thank you for your prayers, for lifting me up when I couldn’t or didn’t
want to do it myself. Thank you for your words of Wisdom and for being there when I
needed you the most, in all ways.
To my wonderful husband Alejandro. Thanks, mi Bebe Precioso, for your loving
gestures, your patience and your presence in my life. You kept and still keep me strong.
You are the perfect partner of Love to share this journey called Life.
To my son Alejandro Jediel and to my future children. Alejandro J., you came to
my life while I was in the middle of my PhD studies and although it wasn’t easy, it was
perfect. You are my principal motivation to fulfill my PhD degree and continue to be
better. I hope your creativity and imagination grow bigger than mine. I know you are
capable of accomplishing those dreams you design.
ACKNOWLEDGMENTS
This isn’t easy. There are so many people to be thankful for that came into my
life and contributed in one way or another. These are the most important, at least, in this
precise moment.
To the Energy of the Universe that has so many names in different
denominations. I acknowledge and thank you for your Presence and for all the Miracles
that were manifested in this process of becoming a more awesome soul.
To my Angels. You know who you are. I acknowledge you all wherever you are.
Thanks for listening and for easing my mind and my soul when times seemed difficult to
continue.
To my whole family, Mami, Papi, mis hermanos Robertito y José Gabriel, mi
esposo Alejandro, mi hijo Alejandro Jediel, mis Suegros y Cuñados. I acknowledge your
presence, your calls, your thoughts, and your good wishes. All of these served as
Energy to accomplish this great journey. I will carry them in my heart for ever and ever.
To my friends, my extended family. I acknowledge your support and kind words
of motivation.
To my labmates. I acknowledge you. How could I finish this adventure without
you? Brianna, Michelle, Thomas, Je-In, Indu, Pingyan, Rupal, Sri, Lily, Vinit, Matthew,
Anna, Cristina, Hao, Wei, Jie, Alex, David, Nicole and Emily. You all are incredible and
amazing characters. No one had better labmates than me and is lucky enough to call
them friends. I learned so much from you, all of which I treasure in my heart. I thank all
of you for your support, motivation, experimental and non-experimental ideas and
conversations, and good times. Michelle and Brianna, thanks for being part of my life in
all aspects as true partners in friendship and sisterhood. You ladies are my
accomplices.
To my amazing mentor, Dr. Dmitry I. Gabrilovich. I want to thank you first, for
allowing me to be a part of your amazing laboratory. Thanks for sharing your knowledge
and laboratory experience with me. Thanks for your guidance in my research training
and for entrusting me with such an exceptional and challenging project, which I feel
very, very proud of. Thanks for showing me how to do great Science. Thank you for
being so patient and understanding of me while I gained experience as a graduate
student during research time and also in many personal situations. I couldn’t ask for a
better and more brilliant mentor. I admire you immensely. You’ll always be my Boss,
Dr. G.
To my committee members, Dr. Julie Djeu, Dr. Esteban Celis, Dr. Thomas Klein,
Dr. Peter Medveczky and Dr. Tomar Ghansah. Thank you for your very meaningful
advice and for challenging me to become a better scientific thinker. Thanks for your
support and encouragement. Dr. Celis, thank you for allowing me to become a nonofficial member of your lab during my last months at Moffitt Cancer Center. That
opportunity allowed me to finish the last experiments and start the writing process.
To Dr. Ghansah. I want to express special gratitude to you for becoming part of
my committee in such short notice. I thank you for accepting and making time in your
busy schedule to evaluate my work.
To my external chair, Dr. José R. Conejo-García. I thank you for accepting my
invitation and taking the time to be part of my dissertation committee as the External
Chair.
To the Department of Molecular Medicine, at the College of Medicine, USF. I
really acknowledge you. I would like to express my gratitude to Dr. Robert Deschenes,
Dana, Michael, Andrew and Kathy Zahn. Thank you for everything that you have done
to make this possible. Dr. Deschenes, thanks for your availability and for what you do in
favor of graduate students. Personally, thank you for your advices and support.
TABLE OF CONTENTS
LIST OF FIGURES .................................................................................... iv
ABSTRACT ............................................................................................... vi
CHAPTER 1. INTRODUCTION .................................................................. 1
Inflammation induces Cancer ............................................................ 1
Carcinogenesis .................................................................................. 9
Models of Carcinogenesis ...................................................... 10
DMBA and TPA model .................................................. 12
TgAC mouse model ....................................................... 13
Urethane and Smoking model ....................................... 15
Myeloid cells involved in tumor promotion........................................ 19
Tumor Associated Macrophages (TAM) ................................. 25
Myeloid-Derived Suppressor Cells (MDSC) ............................ 27
Tumor Associated Neutrophils (TAN) and their
relationship with PMN-MDSC ............................................. 30
S100A9 and S100A8 Proteins ................................................ 34
Adaptive Immune System contributes to Inflammation
induce Cancer ............................................................................. 37
The development and expansion of T lymphocytes ......................... 38
T cells presence in Chronic Inflammation and Cancer ..................... 40
T helper 1 (Th1) and T helper 2 (Th2) cells............................. 42
T helper 17 (Th17) cells .......................................................... 45
T regulatory (Tregs) cells ........................................................ 50
Chemokines and Inflammation ........................................................ 51
Macrophage Inflammatory Protein-1 beta (MIP-1β)/ CCL4 ..... 55
Myeloid cells induce T cell migration towards the inflammatory
site .............................................................................................. 58
Tumor cells need Inflammation for survival and metastasis ............. 60
Hypothesis ...................................................................................... 63
Aim #1 .................................................................................... 64
Aim #2 .................................................................................... 64
i
CHAPTER 2. IMMATURE MYELOID CELLS FOSTER TUMOR
DEVELOPMENT VIA RECRUITMENT OF CD4+ T CELLS ................. 65
Rationale ......................................................................................... 65
Results ............................................................................................ 67
The role of immature myeloid cells in tumor development ...... 67
Accumulation of in skin IMC did not result in immune
suppression........................................................................ 71
IMC recruit CD4+ T cells to the skin ....................................... 78
The mechanism of IMC inducible recruitment of
CD4+ T cells ...................................................................... 82
Discussion ...................................................................................... 85
Methods ........................................................................................... 91
Mice ........................................................................................ 91
Skin carcinogenesis ................................................................ 92
Tissue preparation and histology ............................................ 93
Cytokine expression measured by qPCR and ELISA.............. 93
Western Blot ........................................................................... 94
Experiment with depletion of cells and neutralization of
chemokine in vivo............................................................... 95
CD4+ T cell polarization experiments ..................................... 95
Skin cell isolation and analysis ............................................... 95
Chemotaxis ............................................................................ 96
Langerhans cell staining ......................................................... 97
DC migration and T cell proliferation ....................................... 97
Statistical Analysis .................................................................. 98
CHAPTER 3. MYELOID-DERIVED SUPPRESSOR CELLS IN THE
DEVELOPMENT OF LUNG CANCER ................................................. 99
Rationale ......................................................................................... 99
Results .......................................................................................... 100
Exposure of mice to cigarette smoke induces expansion
of myeloid cells in mice .................................................... 100
MDSC are critically important for tumor progression ............. 103
Discussion .................................................................................... 104
Methods ......................................................................................... 108
Mice ...................................................................................... 108
Lung carcinogenesis model .................................................. 108
Preparation of single cell suspension and antibody
staining ............................................................................ 109
T cell suppressive activity of MDSC ...................................... 109
ii
Evaluation of lung tumors ..................................................... 110
Evaluation of myeloid cells by flow cytometry ....................... 110
Statistical analysis ................................................................ 111
OVERALL CONCLUSION AND FUTURE THOUGHTS .......................... 112
REFERENCES CITED............................................................................ 121
ABOUT THE AUTHOR ............................................................... END PAGE
iii
LIST OF FIGURES
Figure 1.
Scheme of carcinogenesis model where cells undergo
neoplastic transformation in stage-dependent manner ........... 11
Figure 2.
Structure of transgene plasmid construct used to generate
TgAC transgene mouse line by pronuclear injection ............... 15
Figure 3.
Vector used for generation of S100A9tg mice by
microinjection to fertilized FVB/N zygotes ............................... 35
Figure 4.
Expression of S100A9 protein in different organs of WT
and S100A9tg mice ................................................................ 68
Figure 5.
The presence of myeloid cells in the skin of WT and
S100A9tg mice ....................................................................... 69
Figure 6.
Myeloid cells accumulation in the bone marrow after TPA
treatment of mice skin............................................................. 72
Figure 7.
IMC exacerbate papilloma formation ...................................... 73
Figure 8.
Deficiency of IMC resulted in less papilloma formation ........... 74
Figure 9.
Accumulative IMC are not suppressive ................................... 77
Figure 10. Characterization of DC in the skin of TPA treated mice .......... 80
Figure 11. IMC recruit CD4+ T cells to the skin ....................................... 81
Figure 12. Characterization of T cell from skin of S100A9tg and
WT mice ................................................................................. 83
Figure 13. Activated IMC induce CD4+ T cells migration ......................... 84
iv
Figure 14. CCL4 is responsible for the recruitment of CD4+ T cells
to the skin ............................................................................... 86
Figure 15. IMC effect on papilloma formation is mediated via CCL4 ........ 89
Figure 16. Illustration of potential mechanism of IMC to induce tumor
development ........................................................................... 92
Figure 17. The proportion of myeloid cells in peripheral blood of mice
exposed to CS ...................................................................... 101
Figure 18. The proportion of myeloid cells in different organs of mice
exposed to CS ...................................................................... 102
Figure 19. Gr1+ CD11b+ myeloid cells from mice exposed to CS
do not suppress T cell proliferation ....................................... 103
Figure 20. The effect of S100A9 deletion on tumor progression in
mice exposed to urethane and CS ........................................ 105
v
ABSTRACT
Although there is ample evidence linking chronic inflammation with cancer, the
cellular mechanisms involved in early events leading to tumor development remain
unclear. Myeloid cells are an intricate part of inflammation. They consist of mature cells
represented by macrophages, dendritic cells and granulocytes and a population of
Immature Myeloid Cells (IMC), which in healthy individuals are cells in transition to
mature cells. There is a substantial expansion of IMC in cancer and many other
pathological conditions which is associated with pathologic activation of these cells. As
a result, these cells acquire the ability to suppress immune responses and are termed
Myeloid-derived Suppressor Cells (MDSCs). Although the role of MDSC in immune
suppression in cancer and tumor progression is well established, their contribution to
tumor development is still uncertain. The fact that cells with MDSC phenotype and
function are observed in chronic inflammation raised the possibility that these cells can
contribute to initial stages of tumor development. To address this question, we used an
experimental system where the number of IMC was regulated by the expression of
S100A9 protein.
In this project, we used two different models of chronic inflammation in S100A9
transgenic (S100A9tg) and S100A9 knock-out (S100A9KO) mice. In the first model, we
created the conditions for topical accumulation of these cells in the skin in the absence
of infection or tissue damage using S100A9tg mice. Accumulation of IMC in the skin
vi
resulted in a dramatic increase in the formation of skin tumors during epidermal
carcinogenesis. Conversely, lack of myeloid cell accumulation in S100A9KO mice
substantially reduced the formation of skin papillomas. The effect of IMC was not
associated with immune suppression but with the recruitment of CD4 + T cells mediated
by CCL4 chemokine released by activated IMC. Elimination of CD4 + T cells or blockade
of CCL4 abrogated the increase in tumor formation caused by myeloid cells. Thus, this
study implicates the accumulation of IMC as an initial step in facilitating of tumor
formation, which can mediate the recruitment of CD4+ T cells via the release of CCL4
chemokine.
In the second model, we used inflammation-associated lung cancer caused by
the chemical lung carcinogen urethane in combination with exposure to cigarette smoke
referred to throughout as CS. Exposure of mice to CS alone resulted in a significant
accumulation of cells with typical MDSC phenotype in different organs; however, these
cells lacked immune suppressive activity and could not be defined as bona fide MDSC.
When CS was combined with the single dose of urethane, it led to the accumulation of
immune suppressive cells. The expansion of MDSC followed the onset of lung tumors
development. This suggests that MDSC in this model is not the preceding factor but
rather a consequence of tumor formation. Further studies are necessary to determine
the relevance of targeting these cells for cancer treatment and prevention.
vii
CHAPTER 1. INTRODUCTION
Inflammation induces Cancer
Inflammation has been described as a defensive natural reaction towards an
infiltration of a foreign agent and is characterized by swelling, pain, redness and loss of
function of the involved tissue.1 The first job of the immune system is to maintain
homeostasis; to protect the host from the invasion of a foreign agent and to discard any
damaged cells.
Acute inflammation is the first line of defense in the mammalian
immune system.2 Right after an intruder invades the tissue, leukocytes enter to the
vicinity and start secreting oxidative factors, cytokines and chemokines to kill the
pathogen. After the environment is being cleared-up from the pathogen, healing and
repair processes start with secretion of anti-inflammatory factors and apoptosis of
inflammatory cells.
This process occurs in a relatively short period, from hours to
several days.2-4 Conversely, chronic inflammation occurs when there is no resolution of
this first onset, resulting in long-lasting effects.5
The cellular characteristic of chronic
inflammation is the constant presence of macrophages, granulocytes, and other
inflammatory cells that continuously secrete oxidative factors, cytokines, and
chemokines causing DNA and tissue damage.
This persistent inflammatory
environment can develop into neoplasia by the sustaining cell proliferation and inhibiting
apoptosis pathways.
The critical participation of chronic inflammation has been
1
identified in different diseases including Alzheimer’s disease, arthritis, diabetes,
cardiovascular diseases pulmonary diseases and cancer.6
In the mid-1800s, a German scientist, Dr. Rudolf Virchow, observed
“lymphoreticular infiltration” in neoplastic tissue and suggested a correlation between
inflammation and tumor development.7 But it was only in the past 15 years that studies
could prove the participation of inflammation in carcinogenesis and further clarify that
the inflammatory microenvironment provides the essential ingredients for the
progression of tumors.8-10 The distinctive characteristic of cancer-related inflammation is
the incorporation of inflammatory cells, tumor cells, stroma, and inflammatory mediators
at the site of injury with the participation of tissue remodeling and angiogenesis.10,11
The connection of inflammation and tumor development can be viewed in two
pathways, extrinsic and intrinsic. In the extrinsic pathway, environmental factors such
as microbes and viruses can initiate the pathologic process in tissues by sensitizing the
neighboring environment to cancer development. When an infection occurs, the healthy
immune system recruits leukocytes to the affected area to start eradicating the foreign
agent.
When this process becomes chronic, it increases the risk of cancer
development.
5,12
Conversely, in the intrinsic pathway, the neoplastic process starts
with the activation of oncogenes, chromosomal rearrangement, and/or down-regulation
of tumor suppressor genes. Other significant changes in the DNA, such as aberrant
modification of histones, abnormal DNA methylation and deficiencies in DNA repair, can
also lead to cancer initiation. 13,14
A 2008 Harvard study determined that 16-18% of new cancer cases are
attributable to preventable infections.15,16 Among infections, agents that can induce
2
cancer are Hepatitis B virus (HBV) and Hepatitis C virus (HCV), Human Papillomavirus
(HPV) and bacterial strains from Helicobacter species.17 An inflammatory environment
coordinates host responses against microbial infection and mediates tissue repair and
regeneration; however, these responses can have negative impacts when the
inflammation is not resolved in a timely manner.9 This is the case of bacterial infection,
such as Helicobacter pylori.
Infection with H. pylori can generate inflammatory
conditions, such as gastritis, which it is consider a very important factor in the
development of stomach cancers.12,18,19 This inflammation consisted of the infiltration of
leukocytes that release reactive oxygen species (ROS) and reactive nitrogen species
(RNS) among other genotoxic molecules.20 Generally, these molecules are potent antimicrobial agents but can cause damage of DNA and DNA repair mechanisms in the
infected and neighboring cells.
In order to restructure the damaged tissue, the
inflammatory environment can provide survival and proliferative signals to the “initiated”
cells leading to carcinogenesis.9 Moreover, H. pylori can secrete the products of
cytotoxic-associated gene A (CagA) into gastric epithelial tissue, which can then cause
gastroduodenal mucosal inflammation, atrophic gastritis and gastric carcinoma.21 H.
pylori perhaps represent one of the most studied bacteria that induce chronic
inflammation that leads to cancer development. However, other “microorganisms”, such
as viruses, are arising as serious promoters of cancer development. Within the past two
decades many studies exposed the close link of virus infection and its effect on cancer
onset.
Viruses represent another group of infectious agents that can produce
inflammation which leads to cancer. Virus infection represents an estimated 15 percent
3
of all cancer burdens.22 Infectious agents such as viruses can exert molecular
mechanisms which lead to prevent apoptosis and uncontrollable proliferation.23 Some
latent viruses can transform the cell into a malignant one.24 Viruses employ different
ways to remain persistent and lead to carcinogenesis. One way is the activation of
immunosuppressive mechanism through regulatory T cells (Tregs), which downregulate the acute inflammatory responses to kill the pathogen.25 Immunosuppressive
mechanisms allow the host to become susceptible to other infections and to keep the
virus inside the cells.25,26 The mechanism by which the viruses utilize cell transformation
depends on its own genetic material. Viruses with DNA material can persist by
integrating their genetic material into the host genome. This “new” genetic information
can target tumor suppressor genes (p53, pRB) that are essential for making virus
copies and cellular transformation.27
With RNA cancer viruses, like Human
Immunodeficiency Virus (HIV) and human T cell-lymphotropic virus type 1 (HTLV1), the
genetic material is converted into DNA first and then integrates close to an oncogene in
the host genome. This integration can lead to overexpression of the oncogene, which
promotes over-growth of the cell.23,24,28
Other RNA viruses, such as HBV and HCV,
cannot integrate into the host DNA efficiently and instead accomplish their tumorgenic
ability by inducing chronic inflammation in the liver.23 This harsh environment fosters
mechanisms and tools that can alter the DNA of the epithelial cells and thus contribute
to malignant transformation.29
It has also been suggested that our own microbiota, which is helpful for
preventing diseases, can contribute to the initiation of inflammation-induced cancer.30
For example, in 2009, Uronis et al. studied the contribution of host microbiota in the
4
development of colitis-associated colon cancer (CAC). They treated the susceptible IL10 knock-out (IL10KO) mice with azoxymethane (AOM) carcinogen as a colon tumor
initiator followed infecting with colitogenic bacteria Bacteroides vulgatus to trigger
chronic colitis. They found that AOM-IL-10KO mice treated with Bacterides vulgatus
microbiota induced severe chronic colitis developed CAC, as compared to germ-free
counterparts that remained disease-free. This is supported by the additional finding that
MyD88 signaling, and important transcription factor for toll-like receptor (TLRs)
expression, is involved in the development of tumor lesions in IL-10KO mice.
In this
particular study the bacterial infection that cause inflammation can trigger early
malignant changes induced by a carcinogen, which can help the establishment of
uncontrolled cellular proliferation. In summary of this particular study, the presence of
colitis is directly related to the proportion of tumor multiplicity and may act as tumor
promoter.31 In another study that uses the same susceptible mice IL-10KO, Arthur et al.
suggested that commensal bacteria Escherichia coli (E. coli) induce the development of
carcinoma in the gut upon carcinogen treatment into these mice. The authors found that
bacterial products promote tumor appearance by inducing inflammation. However,
alteration of bacterial products could decrease tumor counts but not the inflammation
related to the infection. Therefore, the inflammation associated to colitis can increase
the tumor appearance by altering microbial composition and increase the number of
microorganisms that has genotoxic capabilities.32 These two studies provide strong
evidence of the contribution of bacteria in carcinogenesis through the development of
inflammation. 31,32
5
Contrary to the promoting effects that the gut microbial population has in tumor
formation other studies showed that the same population can be necessary for a
successful anti-cancer treatment. Recently, it was shown that commensal microbiota in
the intestine environment can influence the outcome of tumor-targeted therapy. Iida et
al. showed that the therapeutic effect of combination of CpG-Oligonucleotide and
interleukin-10 receptor (IL-10R) antibody was negatively affected by the absence or
decrease of bacterial load in the gut in tumor-bearing mice. The antibiotics significantly
decreased the presence of tumor-myeloid cells and its inflammatory cytokines such as
TNF, thus promoting tumor growth. Apparently, when mice are treated with anti-tumor
therapy the commensal microbiota stimulate the tumor-infiltrating leukocytes to produce
cytokines that negatively affect tumor growth. Further, they demonstrate that antibiotics
can also have a negative effect in cancer chemotherapy. They used a platinum
compound, oxaliplatin, which form DNA adduct and induces ROS production and
cytotoxic T cells immunity.33,34
Treatment with antibiotic reduces the effect of the
oxalipatin and decrease survival of tumor-bearing mice. Since ROS production is
recognized in myeloid cells they depleted Gr1+ cells and found that the effect of
oxilipatin was impaired in tumor bearing mice.
These data suggest that antibiotics
could also affect the ROS production by the tumor-myeloid cells in tumor-bearing
mice.35
Following the same idea, another study showed how antibiotics alter the effect
cyclophosphamide (CMX), a chemotherapy drug that induces tumor cell death by
regulating adaptive immune responses. CMX treatment in tumor-bearing mice caused a
dysregulation of the intestinal barrier tissue and microbial population which induce a
6
mobilization of local Gram positive- bacteria to lymphoid organs. This dysregulation
caused a decrease of RORgt+CD3+ T cells and CD103+CD11b+ DCs in the gut
environment. Further, in an in-vitro experiment they could differentiate the CD4+ T cells
found in the spleen into Th17 and Th1 cells upon CMX treatment. Introduction of
antibiotic into these CMX-treated mice failed to differentiate the naive CD4+ T cells into
Th17 or Th1. This result showed the importance of the gut microbiota in the
differentiation of T helper cells by chemotherapy treatment. Moreover, CMX was able to
induce the pathogenic Th17 (pTh17) within the spleen in the presence of gut microbiota
which helped decrease tumor burden. Gram-positive antibiotic in tumor bearing mice
inhibited the anti-cancer effect of CMX thus the presence of splenic Th1 and Th17 in
CMX-treated mice. Interestingly, they performed an adoptive transfer of in-vitro
developed pTh17 into antibiotic-treated mice and showed that CMX could recapture its
anti-cancer effect.36 These two studies recapitulate the importance of the host natural
resources, such as commensal bacteria, in the eradication of cancer and highlighted the
participation of activated leukocytes in the eradication of established tumors.
As suggested previously, environmental factors can also influence the
development of chronic inflammation. It is well known that tobacco and its derivatives
can accelerate lung cancer development.37 Studies have shown that inflammation
caused by tobacco-associated materials can promote cancer in mice. For instance,
Takahashi et al. demonstrated that after exposing mice to tobacco smoke, there was a
significant increase in leukocytes population and in cytokines and chemokines mRNAs
in the inflamed lung environment.38 Other environmental factors can also induce cancerpromoting inflammation without directly causing genetic alterations. For example, during
7
the phagocytosis of particulates, such as asbestos and silica, reactive oxygen species
(ROS) are secreted and can activate inflammasomes, a large cytoplasmic protein
complexes39, which triggers the recruitment of inflammatory leukocytes and cytokines in
the lung.40
Obesity, one of the major diseases of our century, has been proven to contribute
to tumor development as well.41
Many studies suggest that obese and overweight
populations have an increased incidence in the development of different types of
cancer. Obesity not only influences the tumor progression, but it can affect treatment
outcomes, leading to an increase in mortality.42
Obesity is known to be closely
associated with a chronic state of inflammation. Over-weight individuals secrete more
pro-inflammatory factors, such as TNF-α, IL-6, inducible nitric oxide synthase (iNOS),
TGF-β, etc., than slimmer individuals. As the adipose tissue expands, the inflammatory
mediators
can
affect
the
homeostasis
of
the
surrounding
normal
tissue.43
Macrophages, present in adipose tissue of obese individuals, are responsible for the
secretion of these pro-inflammatory mediators.43,44 Moreover, adipocytes, the cells that
compose the adipose tissue, can enlarge, induce hypoxia, and secrete more
inflammatory cytokines and chemokines that contribute to developing the inflammatory
environment.45 Another study examined the infiltration of CD8+ T cells into the obese
adipose tissue and presented data suggesting that these cells can also regulate
inflammation.45
It is suggested that all of these factors, which contribute to the
development of chronic inflammation in adipose tissues, also predispose the affected
cells to DNA instability and activation of oncogenes; factors necessary for the initiation
of cancer. 46
8
It is now becoming evident that many of the factors that contribute to the chronic
inflammation may lead to cancer development primarily by altering the intracellular and
tissue environment to become conducive for carcinogenesis.
Carcinogenesis
Cancer is one of the leading causes of death in the United States.47
Although
research has contributed to the early diagnosis, treatment, and prevention of cancer,
there is still a high incidence of new cases.48 As mentioned above, epidemiological and
laboratory studies completed in the past decade strongly support the association of
chronic inflammation with the onset and progression of malignancy.49,50 Carcinogenesis
is the process by which a normal cell transforms to a malignant cell by a series of
genetic mutations. Rackoff-Nahoum described carcinogenesis as possessing six
fundamental properties: (1) self-sufficient proliferation, (2) insensitivity to antiproliferative signals (3) evasion of apoptosis, (4) unlimited replicative potential (5) the
maintenance of vascularization and, for malignancy, (6) tissue invasion and
metastasis.9 To better assess the characteristics and function of cells in the neoplastic
environment, these properties have been grouped into 3 phases: initiation, promotion,
and progression.51
An important event for the development of cancer is the
accumulation of genetic mutations in the specific tissue. These modifications in the
genetic materials are not only required in the initiation stage, but also are favorable in
the subsequent progressive stages.9
9
Models of Carcinogenesis. Cancer can originate from any part of the body.
The multistage structure of tumor formation, which can be adapted to most types of
cancer, arises from different tissues.51 For many decades, the induction of tumors in
mice has been used to study the development of cancer. It is understood that cancer
has both environmental and genetic etiologies. Mice models are a very useful and
controllable tool for studying these two factors independently.52
Carcinogenesis is
generally described as a multiple-stage phase in which a healthy cell is converted to a
cancer cell via a series of mutations. When a normal cell undergoes the first mutation, it
is called “initiation.” If this mutation affects the expression or activation of a protein that
promotes growth, this cell will experience accelerated growth and will enter into a series
of intermediate states, including a pre-neoplastic stage.53 Subsequently, a selective
clone from the neoplastic cell may experience more mutations and consequently be
transformed into a malignant tumor cell. This new malignant tumor cell can secrete
factors that influence the environment and induce the other neighboring cells to go thru
the same transformations.52,54
The following stage is called “promotion” stage. During the promotion stage,
various chemicals (tumor promoters) support cell proliferation and inhibition of
apoptosis. Additionally, this stage is characterized by chronic inflammation. 55,56 Tumorpromoting agents are non-carcinogenic substance that are capable of activating the
recruitment of leukocytes, which contribute by secreting growth factors, chemokines and
cytokines that promote angiogenesis, more cell proliferation, and suppress host
immunity. Inflammatory cells also produce ROS that can promote further mutations in
the transformed cells. All of these events promote malignant transformation, clonal
10
expansion and progression of cancer.3,56 This multi-stage model of carcinogenesis
allows for distinction between the initiation and promotion stages of carcinogenesis,
which helps the analysis of the process.57 In the past decades different methods have
been developed to study the carcinogenesis. Most of these models have in common the
use of an initiator, a carcinogen, and a tumor promoter, a non-carcinogen agent which
are essential elements in the carcinogenesis process (Figure 1).
Figure 1. Scheme of carcinogenesis model where cells undergo neoplastic
transformation in stage-dependent manner.
11
DMBA and TPA model. In experiments designed to study multistage
carcinogenesis, the initiation stage is triggered by a carcinogen or an activated
oncogene. 7, 12-dimethylbenz(a)anthracene (DMBA) is the most common topical
carcinogen used in studies examining the biological process of carcinogenesis.
Although this event is permanent and irreversible no tumors will grow until there is a
promotion environment, such as the recruitment of inflammatory cells. DMBA induces
mutations by creating metabolites that covalently attached to DNA forming DNA
adducts.58 The main target of this carcinogen is H-ras oncogene. H-ras is located in
chromosome 7, in an invariable site, which favored the strong selection for the mutant
copy.59
DMBA cause a mutation in codon 61 of the H-ras gene changing the adenine
for a thymine which form DNA adducts (chemical carcinogen covalently bond to the
DNA), known to cause tumorgenesis.52,60
Besides H-ras oncogene, DMBA has been
shown to cause mutations in other members of the Ras family. Studies made in H-ras
KO mice with DMBA treatment showed fewer papilloma development in these mice
compared to WT counterparts suggesting the involvement of other Ras genes such as
K-ras and N-ras.61
After establishing the initiation stage, the transformed cells expand clonally
during the promotion stage.62 Tumor promotion can be instigated by the repeated
application of a tumor promotion agent within the vicinity of the original application of
carcinogen. The promotion stage is characterized by the onset of chronic inflammation
and the eventual cancer development. In this phase the mutated cells increase
proliferation and decrease differentiation and apoptosis.63 Tumor promotion can be
instigated by the repeated application of a chemical agent in the vicinity of original
12
application of carcinogen. The classical and most popular skin tumor promoter is 12-Otetradecanoylphorbol-13-acetate (TPA) also known as phorbol 12-myristate 13-acetate
(PMA). UV light can also elicit the promotion phase. The resulting clonal expansion of
the initiated cells cause the hyperplasia of the epidermis.64 TPA is one of the most
potent phorbol diester that activates signaling thru Protein Kinase C (PKC) enzyme,
which is a calcium activated phospholipid-dependent serine/threonine kinase.65
Activation of PKC allows phosphorylation of transcription factors that induce the
constant activation of oncogenes such as Ras.66 Several PKC isoforms are crucial for
the establishment of inflammation and the increased proliferation of the malignant cell
that are necessary for tumor development.
67,68
These chemical reagents are the most
used in experiments to study the carcinogenesis induction and progression in animals
that don’t harbor any mutation in the Ras gene family. However, there are other animal
models that facilitate the studies by having the alteration in the genome that are
expressed as mutation and are considered the initiation stage in the carcinogenesis
model. FVBN and TgAC are mice model widely studied and use to perform experiments
to search for information about the process of carcinogenesis. Generally, mouse model
provide a more realistic insight about how the progress of a mutation can lead to tumor
formation.
TgAC mouse model. Among the carcinogenesis multi-stage models TgAC mice
represent one of the best studied and the most employed transgenic mouse model for
learning about tumor progression in a stepwise fashion.69 This animal model is used to
13
develop strategies for cancer prevention and to identify specific characteristic of the
particular environment during tumor initiation, promotion and progression. Interestingly,
these mice were originally created for hematopoietic studies but a particular founder
line, TgAC ( as used in the studies described in this dissertation), developed epidermal
papillomas where traumas were inflicted.70 This observation was significant because it
provided strong support for previous studies that already suggested a correlation
between chronic irritation and wound repair with the early stages of carcinogenesis.
These mice carry an altered oncogene that contributes to tumor progression in other
animal models and human malignancies.71 Specifically, TgAC transgenic mice contain
inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin
to these animals.70-72 The 5’ end of this oncogene is fused to an embryonic Z-globin
promoter which is transcriptionally activating response to wounding, UV radiation and/or
specific chemical, thereby increasing expression of H-ras and promoting the
development of skin papillomas (Figure 2). In addition, in the 3’ end this construct has a
DNA segment encoding SV40 splice/polyadenylylation which provides both RNAse
protection and a DNA probe for identification of the transgenic mice.
70
Hence, the
TgAC mouse line is considered an appropriate alternative to recreate and study in detail
the carcinogenesis process since many human cancers have high incidence of
mutations in RAS gene family. 73,74
To achieve tumor promotion it is necessary to apply repeatedly the chemical
agents, such as TPA, which led to hyperplasia in the epidermal area. Also exposure to
UV light and scratches from cage mates can lead to overgrowth of the skin in these
mice. These tumor promoting activities can stimulate production of growth factors,
14
Figure 2. Structure of transgene plasmid construct used to generate TgAC
transgene mouse line by pronuclear injection 71
mobilization of cytokines and chemokines, and generation of a hypoxic environment
which is associated with tissue inflammation.
Throughout the tumor promotion stage there is inflammatory cell infiltration,
increased DNA synthesis, and proliferation of basal keratinocytes.75 During epidermal
hyperplasia the group of mutated cells have a growth advantage that allows the
selective expansion.75 The final step of the promotion stage is the development of clonal
outgrowth known as papilloma.76
Urethane and Smoking model. Lung cancer is one the deadliest types of cancer.
Since the early 60’s, the causal relationship between smoking and lung cancer was
established.24 Further, it become clear that exposure to second hand smoke increases
15
the chance of non-smokers to develop lung cancer.24 Also, the use of tobacco has been
shown to be a significant contributor in the development of several other lung diseases
related to consumption of tobacco. Chronic obstructive pulmonary disease (COPD),
also known as emphysema or chronic bronchitis, is an inflammatory lung disease that
can be identified with a decline in lung function characterized by a permanent airflow
obstruction and destruction of lung parenchyma due to inflammation.24 Although COPD
is the third cause of death in the United Stated, it is usually preventable and treatable.
Long term exposures to lung irritants, like cigarette smoke, improves the probability of
developing lung cancer along with COPD.77
Therefore, it can be hypothesize that
inflammatory pathways may be shared in the pathogenesis of lung cancer and COPD.
Thus, experimental animal models that recreate the effect of smoking in humans are a
useful tool to study tobacco effects and can further be used to perform pre-clinical tests
of therapy and prevention.78 Moreover, this type of models can be used to analyze the
health risks associated with exposure to environmental tobacco smoke by integrating
epidemiological studies with results derived from animal experiments.79
In most cases, murine tumors share morphological, histopathological and
molecular characteristics with human tumors. Murine pulmonary adenomas are wellcharacterized pre tumorous lung lesions that show histological similarities to human
non-small cell adenocarcinomas. These murine lung pre-tumorous lesions are the
immediate precursors of malignant lung adenocarcinomas in mice.78,80 The majority of
lung adenoma/adenocarcinoma experiments utilize single intraperitoneal injection of
lung chemical carcinogen in a susceptible strain such as A/J mice.78 Chemical
16
carcinogens are a very useful tool to induce lung tumors in a reproducible and
consistent way.
An ample array of chemicals with different potency can induce
carcinogenesis of the lungs in mice including urethane, smoke components (polycyclic
aromatic hydrocarbons and nitrosamines), metals and aflatoxin.81,82
Among the
different lung mutagenic initiators urethane is the most common carcinogen used in
murine lung tumorigenesis models. Urethane can be found in tobacco leaves, tobacco
cigarettes and even in fermented food.83,84 Urethane is formed by the reaction of urea
and alcohol.85 Urethane can induce the development of lung tumors in non-ciliated
airway epithelial (Clara) or type II alveolar epithelial cells in mice. After the carcinogen
is introduced via the peritoneum, there is a transient decrease in the proliferation of
these two types of lung epithelial cells– but soon after, they recover the proliferation rate
and exceed the number of cells in control mice.82,86 Some of the genetic alterations
found in the carcinogen induced tumor include the activation of K-ras and the inhibition
of tumor-suppressor genes such as Rb and p53.82,87 The activation of K-ras is the one
of the most common gene mutation found in mice and human lung cancers. In human
lung tumorigenesis, K-ras mutations are detected in high frequency in adenocarcinomas
as well as their precursor stage, suggesting that the mutations within the early stages
are indicative sign of transformation.88 Injecting urethane in AJ mouse strain can cause
an A-T transversion in codon 61 in the K-ras gene. Additionally, in mouse and humans
lung tumors, the tumor suppressor genes Rb and APC were inactivated by methylation
or by mutations.
89
Mutations in p53 were identified in carcinogenesis of the lung
associated with tobacco smoking. However, these mutations are not frequent in the
carcinogen-induce tumors. Studies suggest that mutation in Rb and p53 genes occur at
17
later stages when the tumor is established and ready to progress. These mutations
might happen as a result of DNA damage or genetic instability.87,90
It is now very noticeable that cigarette consumption increases the incidence of
lung cancer dramatically. Even second-hand smokers are at great risk of development
of chronic diseases.
Environmental tobacco smoke (ETS) is a mixture of 85% of
sidestream smoke (SS) and 15% of mainstream smoke (MS). The exposure to ETS
can cause serious health concerns including asthma, bronchitis and even lung cancer
and cardiovascular diseases.91 Initial efforts to cause tumorigenesis using cigarette
smoke in mice indicated that cigarette smoke is poorly tumorigenic in mouse models.
Moreover, early smoke inhalation studies performed in different animal models could
not prove the association of cigarette smoke with the development of lung cancer.92,93
In the past decade Witschi et al. developed a model of cigarette smoking lung
carcinogenesis in the susceptible A/J mouse strain. In his model they exposed A/J mice
to ETS continuously for 5 months followed by 4 months of air exposure or rest.79 The
smoking machine consisted of 2 components. The first one is an automated device for
loading, lightning, and smoking the cigarettes which enabled the mechanical smoking of
up to 10 cigarettes simultaneously. This system uses Federal Trade Commission (FTC)
method of puffing for 2 seconds, at a volume of 35cm 3 every minute. The second
component is a chimney that dilutes, collects and transfers the smoke from the burning
end of the cigarettes to the conditioning chamber. In the conditioning chamber, the
process of mixing and aging of the cigarettes produce a combination of SS and MS. 24,91
Witschi et al. reported that when this combination protocol was performed in the
smoking machine, the incidence and the multiplicity of lung tumor formation increased
18
at least 2 folds after treatment was finished.94,95
They suggested that the period of air
exposure allowed the animals to eat regularly and diminish the metabolic rate which
increased the chances of cancer development. It has been studied that impaired food
intake and high metabolic rate can decrease carcinogenesis risks.96,97 Another
possibility is that ETS can suppress or slow down the process of tumorigenesis in
initiated cells.96
This study allowed us to utilize the information to recreate the
carcinogenesis model with urethane, as the initiation stage carcinogen, and the
exposure to smoke, as the inflammation inducer in the promotion stage. This lung
carcinogenesis model will provide us with a tool to study to the cellular participants
involved in the inflammation phase that can lead to development of lung cancer and
probably other lung diseases.
Myeloid cells involved in tumor promotion
There is a complicated relationship between malignant and immune cells within
the inflammatory environment. Immune cells along with the stromal cells come together
to the damaged site and communicate with each other through direct contact or through
cytokines and chemokines intervention.98
As a result of this “communication” they
perform their functions to restore the normal state of the environment.
However,
sometimes the presence of these activated immune cells, can push the balance towards
the pro-cancerous state.6 Many times these immune cells can greatly contribute in the
generation of tumor stroma, which is defined as group non-malignant cells that can
support tumor progression and metastasis of the transformed cells.99 Members of the
19
myeloid lineage compartment are main participants in the process of tumor
development. In many animal and human models immature myeloid cells can stimulate
tumor
progression
by
supporting
neovascularization,
metastasis
and
immunosuppression.49,100 All these events are part of the inflammation environment that
is created upon the arrival of these cells. Moreover, the type of insult (that is the
mutation or the response of the immune system) within the tissue plays a significant
role in the progress of inflammation into carcinogenesis.2
All this together can
determine the phenotype of the leukocytes that infiltrates into the damaged tissue and
influence the tumor development.
In the inflammatory environment, with the potential of tumor development, a
diverse population of leukocytes has been found, such as neutrophils, mast cells,
eosinophils and macrophages. These mature myeloid cell populations are loaded with
cytokines, chemokines and other soluble factors capable of protecting the host from
invaders.11 Moreover, many studies associate these inflammatory myeloid populations
with the promoting forces of cancer development.
In human studies, chronic
inflammation in the promotion stage is strongly linked to carcinomas in breast, liver,
gastric mucosa, ovary and skin among others.11,101 During tumor promotion and
metastasis, these myeloid cells, along with tumor cells, secrete inflammatory factors
that allow survival, motility and metastasis.102
Possibly the best evidence of
inflammation during neoplastic formation comes from studies in which the use of antiinflammatory drugs, steroids, can ameliorate the cancer risk. There studies suggesting
that that the use of these drugs can reduce colorectal cancer and can help with the
prevention of gastrointestinal cancer development.103-105 A possible explanation of the
20
effect of these drugs is the ability to inhibit the enzymatic activity of cycloxygenases
(COX1 and COX2).106 These enzymes, particularly COX2, are found to be upregulated
in several types of cancers besides the colorectal cancer.103 COX2 can produce
prostaglandin that can feed the inflammatory environment by modulating the immune
system, promoting angiogenesis, regulating cellular invasion and inhibiting apoptosis,
thereby contributing to carcinogenesis.11,107
As mentioned before, within the inflammatory environment a variety of myeloid
and lymphoid cells can be found, which can contribute to tumor development. In healthy
individuals, these cells provide the protection of the organisms against various
pathogens and tissue disruptions. Below, we will discuss some of the most important
cells that are found in the inflammation environment.
Bone marrow-derived cells migrate to the affected tissues in response to
complex of various chemokines. It can amplify the presence of effector cells from
different lineage-committed progenitors and facilitate its mobilization to the circulation.
Many inflammatory cytokines contribute to the proliferation, differentiation and selfrenewal ability of bone marrow cells.108,109 Typically these signals play an important role
in the maintenance of bone marrow cells but when the inflammation becomes
persistent, are these same signals that become a threat to homeostatic environment
and mediate cancer initiation.109
Homeostasis within the bone marrow depends on the replacement of immune
effectors cells by hematopoietic precursors. Hematopoietic Stem Cells (HSCs) are a
rare, multi-potent inactive population present in the bone marrow. They represent only
the 0.01 percent of the total cells in the BM and are capable of replenish the
21
hematopoietic population as they are used or become aged. These cells supply the
progenitors of all differentiated cell types in the blood.110,111
In response to
hematopoietic challenges, such as hemorrhage, chemotherapy and infections, these
HSCs can be activated to proliferate and differentiate.109,112
In the mid 90’s it was
identified 3 groups of hematopoietic stem cells in mice: Long term-HSC, short term-HSC
and Multi Potent Progenitors (MPP).113 Long and Short term stem cells are
characterized by the high expression of markers c-kit, and Sca-1, very low levels of
Thy1.1 and low or negative levels of Lin (lineage) marker. 113 These cells are able to
provide myeloid and erythroid cells necessary for survival in a BM transplantation
experiment.114
Eventually, they start the process of self-renewal to generate more
hematopoietic cells for the rest of the lifetime of the mice. 115 However, the expression of
these surface markers can be altered by inflammatory signals. It was found that Sca-1
is upregulated in the presence of IFNs and TNF116 and c-kit is down regulated during
chemotherapy treatment.117,118 Following this stage, HSC can give rise to multi-potent
progenitor (MPP) cells described with Lin-IL7Rα-Sca-1+c-kit+FLT3low-hiThy1-CD34+
surface markers. These MMPs can differentiate into the common myeloid progenitor
(CMPs)
identified
subsequently
into
with
the
Lin-Sca-1-c-kit+CD34+FcγRII-FcγRIII-
markers
granulocyte/monocyte
progenitors
and
(GMPs,Lin-Sca-1-c-
kit+CD34+FcγRII+FcγRIII+) and megakaryocytes/erythroid progenitors (MEPs, Lin-Sca-1c-kit+CD34-FcγRII-FcγRIII-).111,115 GMPs and MEPs can evolve to become granulocytes,
dendritic cells progenitors, monocytes, as well as inflammatory immature myeloid
cells.119
22
One of the many important transcription factors that regulate the myeloid lineage
differentiation is PU.1. PU.1 is the product of the oncogene SP1 and belongs to E26
transformation-specific (ETS) family of transcription factors. It has been shown that
deficiency of PU.1 affects the cells to HSC regeneration and thus interrupts the eventual
differentiation of CMP to mature cells.120 Interestingly, different amounts of PU.1
regulate the differentiation stages of CMPs and common lymphoid progenitor (CLP)
cells as well as can increase granulopoeisis.121 Even though that PU.1 is required for
most of the early stages of mature myeloid cell differentiation, other transcription factors
influence
greatly
in
later
stages.
Among
these
transcription
factors
are
CCAAT/enhancer binding protein (CEBP’s), Interferon-regulatory factor 8 (IRF-8) and
growth factor independent 1 (GFI-1) which all can activate different genes that influence
the development of chronic inflammation.122
All these transcription factors regulate the expression of cytokines, growth factors
and the receptors that promote the differentiation of myeloid cells. 123
The colony
stimulated factor (CSF) family is the best characterized family of growth factors that are
present during the hematopoietic stages and further they are expressed in later stages
of myeloid differentiation. This family include, among others, granulocyte-macrophage
(GM-CSF), macrophage (M-CSF) and granulocyte (G-CSF) colony-stimulating factors.
They can regulate myeloid cells infiltration and function in healthy individuals, as well as
during inflammation.124 CSFs, particularly M-CSF and GM-CSF, are upregulated in the
site of inflammation and are closely related to tumor establishment, progression, and
invasion.
The function of these CSFs is not limited to support the inflammatory
environment; they also contribute to the recruitment and proliferation of inflammatory
23
cells that sustain carcinogenesis. During the development of inflammation these CSFs
can act as cytokines allowing communication between the myeloid cells lineage, stroma
cells and the nearby cells promoting the establishment of transformed cells.124 CSFs
can also influence the plasticity of macrophages in the tumor environment.
For
example, in an in vitro study, GM-CSF treatment of TAMs with blocked M-CSF signaling
resulted in expression of proteins that normally are upregulated in DC. 125
It has been shown that GM-SCF can aggravate tumor development. From a
pancreatic cancer mouse model it was exemplified that tumor-derived GM-SCF is
necessary to drive the development of Gr1+CD11b+ cells, which functionally can protect
the tumor from the immune system attack. Neutralization of GM-CSF decreased the
infiltration of Gr1+CD11b+ cells allowing cytotoxic CD8+ T cells to decrease tumor
progression.126 Therapies designed to target these molecules can help at the onset and
during the progression of the tumor.124,127
Myeloid cells play a major role in constructing the environment favoring tumor
progress and invasion.128,129 Tumor promoting agents, such as TPA, can stimulate
enrollment of immature myeloid cells (IMCs) to the site where the carcinogen was
applied. After application of the chemical tumor promoter, IMCs are mobilized from the
BM to the peripheral blood and then recruited to the site where the mutation occurred.
These IMCs are part of the acute inflammatory cells which are the first responders to
the altered site.56,130 Macrophages, granulocytes, mast cells are activated and proceed
to recruit other leukocytes to the invaded tissue and start the process of elimination of
pathogens and wound recovery. In contrast, when homeostasis failed to be restored
these processes remain long lasting and the same myeloid cells can develop alternative
24
functions that promote tumor growth and metastasis.130 As it was discussed before, the
inflammatory cells are imminent participants in the progression of tumor. Elevated
numbers of myeloid cells in the tumor microenvironment area is generally associated
with a poor clinical outcome. Below, we discuss the most relevant information about
these myeloid cells and its relation with the process of carcinogenesis.
Tumor Associated Macrophages (TAM). Tumor associated macrophages are
cells derived from immature monocytes recruited by cytokines and chemokines that are
secreted form the inflammatory environment.129
As the name implies, these
macrophages are found the tumor microenvironment. TAMs can promote tumor growth,
angiogenesis, invasion and metastasis.131 TAMs are a heterogeneous population which
expresses different phenotypes and functions depending on the tumor tissue. Generally
in humans are identified with CD68 surface marker and in mouse with F4/80 surface
marker.99,132
Many studies in cancer patients correlate a high percentage of
macrophages in tumor with poor prognosis. TAMs can influence every stage of cancer
development, that is, initiation, growth and progression.131,133,134
Within the tumor
environment, TAMs can produce potent angiogenic, lymphoagiogenic growth factors
and other chemoattractants that accelerate the neoplastic progression.135 TAMs are
identified in two groups based in the function they performed. The first group are called
M1 - macrophages which are activated by bacterial infections, lypopolysacharide (LPS),
interferon-gamma (IFN-γ) or microbial-induced chemokines such as tumor necrosis
factor alpha (TNF-α).134 These cells can up-regulate major histocompability complex
25
molecule (MHC) in their cell surfaces to increase antigen presentation, and secrete
great amounts of IL-12 and IL-23 which can help elicit effector T cell responses. These
cells release reactive oxygen species (ROS) and nitric oxide species that can eliminate
pathogens as well as elicit anti-tumor cytotoxicity.
56,136
Further, they can secrete
produce great amounts of cytokines that can induce effective adaptive immune
responses.137,138 On the other hand, M2 or “alternatively” activated macrophages, are
induced by the T helper (Th) 2 cytokines, IL-4, IL-10 and IL-13, and can produce
several growth factors that activate the tissue repair process and suppress adaptive
immune responses.139
These cells are found in more protumorgenic hypoxic
environment and can downregulate inflammatory and type 1-immune responses and
can start the process of neoangiogenesis.136,138
Anti-inflammatory cytokines such as
IL-4, IL-13 and regulatory biochemical, such as glucorticoids, can induce polarization of
macrophages towards M2 phenotype and can prevent classic macrophage activation
and function.140,141 Further, tumor cells can help M2 macrophages induce angiogenesis
and immunosuppressive function by upregulating TGF-β, IL-10 and IL-1 receptors.
Therefore, the tumor environment can interfere with the anti-tumor attack from the
immune system and promote invasion and metastasis.141,142 In addition, M2
macrophages can produce matrix metalopeptidase 2 (MMP2) which can contribute to
tumorgenesis and metastasis, in addition to the immunosuppressive chemokines,
including C-C ligand 17 (CCL17), CCL18 and CCL22 that may favor the T regulatory
(Tregs) recruitment.141 Previous studies found that high levels of M2-type macrophages
correlated with an immunosuppressive environment in patients with fibrosarcoma and
ovarian cancer.143 M2-TAMs block anti-tumor immunity by eliminating M1 macrophage
26
mediated innate immune activity and sabotaging cytotoxic T lymphocyte (CTL)
activation. Moreover, these cells are incompetent antigen presenting cells and can
attract Tregs which adds to the immunosuppressive environment.144 This functional
plasticity of macrophages depends on the environment. The presence of TAM is
elevated at late stages of tumors and is associated with poor survival.139,145
Myeloid-Derived Suppressor Cells (MDSC). These cells represent a
heterogeneous population of pathologically activated myeloid cells that includes
precursors of neutrophils, macrophages, DCs, and cells on earlier stages of myeloid
cells differentiation.99,146 It has been shown that these cells can influence the
development of chronic conditions, like autoimmunity, infectious diseases and
cancer.147-149 The first observations of these cells described their accumulation in the
BM and spleens of tumor-bearing mice and peripheral blood of cancer patients.150,151
MDSCs are characterized by high plasticity152-154 described in more details below.
In healthy individuals immature myeloid cells (IMCs) differentiate to functional
macrophages and DCs but in cancer and many chronic diseases, these IMCs can
expand and acquire MDSCs characteristics.148,155 In mice, these cells are defined as
Gr1+CD11b+ cells. Gr1 includes Ly6C and Ly6G epitopes and CD11b, which is a αMintegrin marker of myeloid cells.147,156 In humans, phenotypic identification of MDSC is
more complicated due to the lack of a single marker and the diversity of the cancer
being studied. Despite this, generally, human MDSCs express CD11b and CD33 and
other granulocytic markers with the absence of DC, macrophages and other
27
lymphocytes markers.157,158 Further, two subsets of MDSCs have been identified in
mouse tumor models, characterized by these cells’ different morphology and surface
marker expression.159
One of these subsets are Granulocytic (G-), also known as
polymorphonuclear (PMN-) MDSCs, which have a morphology similar to granulocytes
and monocytic (M-) MDSCs which have its morphology similar to monocytes. G-MDSCs
can be characterized by its expressed markers CD11b+Ly6G+Ly6Clow, and M-MDSCs
expressed CD11b+Ly6G-Ly6Chigh. Additionally, these cells have different suppressive
activities.
G-MDSC uses ROS and little nitric oxide (NO) to facilitate suppressive
functions and M-MDSC up-regulate a series of cytokines but primary use inducible nitric
oxide synthase (iNOS) and arginase to perform its immunosuppressive activity.147,159
Overall, MDSCs are characterized by their potent immunosuppressive activity and the
ability to promote tumor angiogenesis, tumor cell invasion, and metastasis.99 Another
mechanism by which MDSCs execute their suppressive activity against T cells is by
sequestering cysteine an important amino acid for T cell activation. DC and
macrophages are important providers of this essential amino acid. In the presence of
MDSCs cysteine is reduced and is not returned to the environment thus limiting its
availability to T cells.160
Other mechanisms of suppression by these cells include
secretion of TGF-β, the induction of regulatory T cells, down-regulation of L-selectin
expression in T cells and up-regulation of cyclooxygenase and prostaglandin E2.161
MDSCs have been implicated in many types of cancer and now are getting recognition
for its participation during chronic inflammation.162
MDSCs can corrupt immune
surveillance and block the healthy immune system from eradicating newly transformed
cells.
When chronic inflammation is developing, many factors secreted by the
28
inflammatory cells to the environment can promote recruitment and accumulation of
MDSCs from the bone marrow.154,162 Inflammatory mediators such as growth factors
(GM-CSF, G-CSF, VEGF, TGF-β), pro-inflammatory cytokines (IL-1β, IL-6, IL-13, IFN-γ
and TNF-α) and prostaglandin E2 are known to be expressed by mutated cells in
chronic inflammation which in turn can recruit immature myeloid cells.49,162 However, it
has been noted that even in decreased amounts of these inflammatory mediators
MDSCs can be found in the inflammatory environment suggesting that other molecules
might play an important role in the mobilization of MDSCs during inflammation.163 For
example, chemoattractant proteins, such as S100A9 and S100A8, are important
recruiters of MDSCs. Tumor environment and MDSCs itself can secrete these proteins
as heterodimers which bind to the corresponding receptor and cause retention of the
accumulated MDSCs providing a positive feedback for the suppressive function of these
cells.
Moreover, these proteins facilitate the IMCs conversion into MDSCs in the
immunosuppressive environment.163,164 Several molecular mechanisms govern the
participation of MDSCs in pathological conditions.165 Signal transducer and activation of
transcription 3 (STAT3) represents one of the transcription factors that play an important
role in the accumulation, differentiation and function of MDSCs. 165,166 Activation of
STAT3 pathway abrogates differentiation of immature myeloid cells into a mature state
which promotes MDSC accumulation.167 Further, STAT3 is, in part, responsible of the
suppressive capacity of MDSCs. ROS is induced by the transcription of Nox2 which is
directly upregulated by the activation of STAT3.168 Another transcription factor that is
becoming noticeable in the MDSCs accumulation and function is NFκB. Upon ligand
binding to toll-like receptor 4 (TLR-4) there is an activation of NFκB-MyD88 signaling
29
pathway. This is constant with the infiltration of MDSCs in the inflammation and
eventual cancer development since the ligands and molecules that activate the TLRs
are very abundant in this environment. Activation of TLR4 in MDSCs contributes in
tumor progression by stimulating the immunosuppressive environment which prevents
the anti-tumor immunity.169
It is recognizable the importance and the participation of MDSCs in the regulation
of pathological conditions. It is very necessary to clearly understand the molecular
activities that drive the polarization of MDSCs to a suppressive stage. This
understanding will facilitate the development of more accurate therapies in pathological
disorders.
Tumor Associated Neutrophils (TAN) and their relationship with PMNMDSC. Historically, polymorphonuclear neutrophils (PMN) were considered as
peripheral element in tumor progression. Traditionally, PMN’s function was considered
as largely confined to the protection of organism from invading pathogens. PMN
comprise 50-70% of all circulating leukocytes. They are first leukocytes to reach the
damaged tissue.170,171 PMN are characterized by extensive network of different types of
granules that store various proteases and needed to perform antimicrobial functions. 172
PMN are specialized on phagocytosis and killing of the invading microorganisms and
the release of pro-inflammatory factors, such as IL-1β, TNFα and IFNs, defensins,
etc.173,174
30
Tumor stroma can secrete cytokines that promote myelopoiesis and extravasion
of immature neutrophils.175 For example, upon activation of K-Ras oncogene there is
an upregulation of CXC chemokines which cause accumulation of neutrophils and
promotion of tumor growth in the lungs.176
In human cancer, tumor-derived CXCL8,
CCL3, CXCL6 and in murine cancer models CCL3 and CXCL6 are shown to promote
PMN migration through their potent chemokine action.175 Within the malignant tissue
neutrophils can contribute to angiogenesis, cell proliferation and metastasis by secreting
bioactive molecules. Human neutrophils secrete CXCL8 and their murine counterparts
secrete CXCL1 and CXCL2, CXCL10 and CXCL11 and hepatocyte growth factor
(HGF), which promote angiogenesis in different cancers.177,178 Additionally, the
secretion of CXCL8 by PMN contributes to angiogenesis and possibly can induce a
positive feedback for additional neutrophil infiltration within the environment. 177
The existent of two types of tumor infiltrating neutrophils (TAN) in cancer was
suggested as a reflection of the different degrees of activation and that is fully
activated/antitumoral, known as N1 TANs and weakly activated/protumoral, known as
N2 TANs.175,179
N1-type functions include immunoactivating cytokines and
chemokines, lower levels of arginase, a higher capacity of killing tumor cells and
activating cytotoxic T cells (CTL). N2 TANs secretes cytokines, chemokines and other
inflammatory molecules can both help in the tumor progression and attract other
inflammatory cells that strength the tumor establishment and invasiveness. 171,175 One of
the major contributors to this plasticity is TGF-β, which favors the polarization towards
N2 TAN and prevents the generation of N1 TANs.180 TANs and other bone marrowderived cells contribute to tumorgenesis by secreting proteases such as matrix
31
metalloproteinase 9 (MMP-9).181 MMP-9 is involved in various carcinogenesis models
by upregulating secretion of VEGF and promoting angiogenesis.
Participation of neutrophils has been described in several acute diseases like
lung and spinal cord injuries as well as in chronic diseases such as COPD, rheumatoid
arthritis and asthma.170 Recent data have demonstrated that these neutrophils can also
participate further in the inflammation process leading to cancer development.182
Recruitment of TANs into the tumor environment is mediated by chemokines and
cytokines secreted by transformed cells and other cells from the same environment.
The identification of chemokines together with neutrophils within the tumor environment
strongly suggests that these neutrophils are supporting tumor growth and not
performing anti-tumor functions.176,183 In early stages of tumor this cells have been
associated with genetic instability. Further, in the subsequent stages of cancer
progression neutrophils can secrete different molecules that supports the survival of the
tumorous environment.184 In many established human cancers, analysis of peripheral
blood neutrophils counts could help predict the outcome of the disease. But in many
cases this counts needs to be validated with the PNM counts in the tumor site. 182
Introduction of the concept of MDSC in recent years raised the question whether
TAN are in fact PMN-MDSC. These cells share the same phenotype. To elucidate this
Youn et al. investigated PMN-MDSC from tumor-bearing mice and PMN from tumor-free
mice.
In this study they sorted CD11b+Ly6G+Ly6Clow cells from spleens and from
peritoneal cavities after casein-induced mobilization. One of the first differences they
address was in the maturing state. G-MDSC is described among the MDSC population
which by definition they are present in an immature state contrasting neutrophils that
32
are described as fully matured population of myeloid cells. Adding to this description
the scientists found that G-MDSC express higher levels of CD115 (M-CSF receptor)
and CD244 (expressed in NK cells and in some hematopoietic stem cells) in different
tumor models than in neutrophils which these markers are practically absent. These
markers may contribute the characterization of the immature state of G-MDSC versus
neutrophils. In terms of activation when G-MDSCs were expose to LPS these cells
acquired immunosuppressive activity against T cells. However, neutrophils did not show
any immunosuppressive activity, in fact these cells promoted T cell function. Important
to note that in in-vitro studies splenic G-MDSCs in the presence of growth factors can
differentiate into neutrophils within 24 hours. However in an experiment with BrDU they
identified that most of the cells within the tumor environment express CD115 and
CD244 suggesting that tumor-derived factors promote constant influx of G-MDSC and
prevents to differentiate into neutrophils.
suppressive precursors of neutrophils.
This study suggests that G-MDSCs are
Within the tumor environment the possible
differentiation of these precursors’ cells to mature neutrophils is blocked and
alternatively they acquire an immunosuppressive characteristic.178
Generally, myeloid cells populations serve as an important link between innate
and adaptive immunity to counteract invader pathogens as well cancer cells. But also it
has been studied the contribution of these cells in different stages of cancer
development. In addition tumor infiltrating myeloid cells can alter potential anti-cancer
therapy which jeopardizes the patient responses. It is no doubt that future and better
studies of these population are necessary for better characterization and understanding
of
their function not only cancer patients but in individuals carrying other health
33
disparities. Targeting and/or reprogramming these specific populations will improve the
efficacy of cancer therapies and other chronic diseases.
S100A9 and S100A8 Proteins. The role of S100A8/A9 proteins have been
studied in multiple diseases, from acute and chronic inflammation to cancer
development and metastasis.185 S100A9, also known as Calgranulin B or myeloidrelated protein 14 (MRP14), is a member of a large family of S100 Ca-binding proteins
that contains 2EF-hand motifs.186 It can form a functional heterodimer with S100A8
protein (S100A8/A9, calprotectin) and is involved in a diverse intracellular processes,
including arachidonic acid transport, NADPH oxidase activation, cellular signaling
cascades, and cytoskeleton rearrangement.164,186
S100A9 is a pro-inflammatory
cytokine that can translocate into the cytoskeleton of myeloid cells and recruit
leukocytes to the sites of inflammation.185,186
Further these proteins have been
extensively related to cytokine-like and chemokine-like functions in the extracellular
environment. High levels of S100A8/A9 have been detected in many cancers as well as
in chronic diseases.162,187-189 The most notable receptors for S100A9 proteins are Tolllike receptor 4 (TLR4) and receptor for advance glycation end products (RAGE).186,190
Within the inflammatory microenvironment S100A9 is highly expressed by neutrophils
and epithelial cells.187 S100A8/A9 proteins contribute to the infiltration of additional
leukocytes from the circulation, which can enhance the immunosuppressive
environment and potentially facilitate tumor development.154,163,191 Sinha et al.
demonstrated that the tumor environment can induce infiltration of MDSCs by secreting
S100A8/A9 complex. This complex binds to the RAGE receptor present in the immature
34
myeloid cells and signals through the NFκB pathway promoting migration and additional
expression of S100A8/A9 heterodimer by MDSCs.163 Moreover, S100A9 protein has
been linked to defective maturation process of myeloid cells. Upregulation of S100A9
protein is seen at early stages of myeloid lineages differentiation and expression of
CD11b in neutrophils, monocytes and macrophages.186,192. Tumor cells are capable of
interrupting the differentiation of myeloid cells by secreting VEGF, IL-6, M-CSF, and
GM-CSF. In line with these facts, Cheng et al. demonstrated that VEGF upregulated
S100A9 expression in HPC. Transgenic mouse was created where s100a9 was
expressed in hematopoietic cells under control of the H2K-promoter/enhancer and
Moloney MuLV enhancer/poly(A) site.167,193 To trace expression of transgene, GFP
gene was introduced behind an IRES sequence to allow for separate translation.167
(Figure 3)
Figure 3. Vector used for generation of S100A9tg mice by microinjection to
fertilized FVB/N zygotes. 167
Several experiments were performed and suggests that overexpression of this
protein can enhance the progression of tumor in mice. They were able to show that the
overexpression of S100A9 protein accelerates tumor growth. In an in-vitro study HPCs
from S100A9tg (GFP+Tg) mice cultured with LPS generated more Gr1+CD11b+ IMC
than HPCs from WT mice, and significantly decrease differentiation of other myeloid
35
cells phenotypes, CD11c+ and Gr1-F4/80+. These results suggest that upregulation of
S100A9 protein in an inflammatory environment could induce acceleration of tumor
growth and accumulation of IMCs at the expense of differentiated DCs and
macrophages.167
Further, in an in-vivo experiment they showed that over expression of S100A9
not only affected tumor growth, it also increased number of Gr1+ cells in response to
inoculation of mice with tumor cells or CFA. Tumor progression was also studied in the
absence of S100A9. For these experiments they used S100A9KO mice. Manitz et al.
generated these S100A9-deficient mice by replacing the translational start site of the
entire coding region of S100A9 protein with neomycin cassette thus deleting the starting
codon. The absence of this protein deregulates the expression of CD11b in neutrophils
which corresponds to the inability of these cells to response to tumor secreting factors in
vitro.194 In line with these findings Cheng et al. hypothesize the tumor progression is
affected in the absence of S100A9. Indeed, this study demonstrated that the absence of
this protein in tumor-bearing mice can delay tumor progression.
To explain this
phenomenon, they showed that HPC failed to differentiate into DC in the presence of
tumor-cell conditioned media (TCM) from EL4-tumor bearing mice.167
Overall, these results suggest that the bone marrow derived-IMCs that participate
in the tumor development itself are Gr1+CD11+ cells and no other myeloid cells
phenotype. Since early stages of cancer development are characterized by
inflammation with upregulation of molecules, such as S100A9, it is intriguing to
investigate if these same IMCs are required to start the process of tumor formation.
197
195-
During inflammation-induce cancer other cells from the immune system, specifically
36
from the adaptive immune, help to set up the environment for tumor establishment.
These cells, called lymphocytes, can actually suppress the anti-tumor surveillance or
block cytotoxic attack and induce more cytokines that cooperate in the inflammation
process.198-200
Adaptive Immune System contributes to Inflammation induced Cancer
Lymphocytes are critical element of cancer immunosurveillance, capable of
recognizing malignant changes in cells and execute effector mechanisms before the
tumor onset.201 Adaptive immunity is also important for promotion of the inflammatory
environment. An innate immune response in the chronic inflammation activates
effectors lymphocytes via the direct contact with APC. In lymphocytes antigen-specific
receptors are the consequence of random gene rearrangement that allowed a more
extended and flexible repertoire of responses.202,203 In contrast, myeloid cells express
germline-encoded receptors which provide a invariable function.203 The classical
protective function presented by lymphocytes is cytotoxicity via granzyme/perforin
system or engaging death receptor.
204
This effect can be further amplified by the
secretion of pro-Inflammatory cytokines IFNγ and TNFα mainly by T helpers 1 (Th1)
cells.205 Studies characterizing cells during chronic inflammation and tumor formation
revealed the complex heterogeneity of lymphocyte and myeloid nature.201
It is now accepted that the inhibition of adaptive immune system can contribute to
cancer development. This occurrence is mediated by inhibition of CTL, antitumor
chemokines and cytokines, accumulation of regulatory T cells and B cells.206 Animal
37
models experiments provided mixed reports regarding the absence of lymphocytes
during cancer development. For example, in a study of HPV16 cervical carcinogenesis,
elimination of CD4 T lymphocytes resulted in a slight delay on tumor growth; however,
in the same model, female mice treated with estrogen therapy for the elimination of the
CD4 T lymphocyte population resulted in tumor enhancement and predisposition to
cervical carcinoma, as compared to the control group.207 The roles of the immune
system can become hazardous during chronic inflammations as adaptive immune
responses may cause 1) ongoing activation of innate immune system 2) antibody
accumulation resulting in additional recruitment of innate immune cells and 3) T
lymphocyte dysfunction instead of activation.208 Modified T cells and B cells can
regulate aspects of myeloid cells by altering their basic biology which consequently can
control or mismanage the immune response.209 Studies showed that depending on the
tumor environment and the differentiation stage of lymphocytes, these cells have
exchange antitumor and pro-tumor capabilities.210
The development and expansion of T lymphocytes
T lymphocytes undergo several stages of differentiation before they acquire the
mature and/or effector phenotype.210 Lymphopoiesis takes place in the BM and in the
thymus, which are called the central lymphoid tissue.211
Hematopoietic stem cells
(HSC) differentiate to the common lymphocyte progenitors (CLP) but also can give rise
to early T-lineage precursors (ETP). Although CLP can give rise to T cells precursors, it
is the ETP that give rise to the T cell precursor that migrates from the BM to the
thymus.212 Once in the thymus, these precursors received a series of signals from
38
stroma, which include up-regulation of Notch1 receptors. Notch signaling helps early
precursors of T cells to determine the nature of the T cell receptors (TCR) expressing
alpha/beta (αβ) or gamma/delta (γδ) and CD4+ or CD8+ T-cell commitment.213 Before
they acquired mature markers, thymocytes go through several stages of CD4/CD8
double negative cells.
While migrating to the outer cortex of the thymus, they
upregulate CD4 and CD8 molecules. During this double positive (DP) stage, Rag and
TCR genes start to express. Subsequently, the process of differentiation to CD4 or CD8
single positive cells occurs, resulting in the correct assemblage of the TCR and ligand
affinity to the major histocompability complex (MHC) present on APC.214 After
concluding the positive selection, the thymocytes are ready to move to the periphery as
naïve lymphocytes.215 The two major lymphocytes subset, CD4 T cells (or T helper
cells, Th) and CD8 T cells (cytotoxic T lymphocytes, CTL) are activated upon
recognition by their TCR of non-self-antigens presented by MHC class II or MHC class I
respectively.216
Activation of T cell requires essentially two signals that happen at the
same time. Signal 1 is the recognition and engagement of the TCR/CD3 (on the T cell)
with the MHC/antigen (on APC) and signal 2 is the interaction between co-stimulatory
molecules B7 on APC surface with CD28 on T cells surface.217 It has also been also
suggested that a third signal, provided by inflammatory molecules, is necessary for
providing for the maturation of effector T cells.218
219,220
Pape et al. showed that clonal
expansion of CD4 T cells is enhanced by the administration of IL-1β and TNF-α
compared to cells stimulated with antigen only. Previously it wasn’t clear if IL-1β can
directly improve the clonal expansion of CD4 T cells or if was the effect on APC by
upregulating costimulatory molecules.218,221 Later it was demonstrated that signal 3
39
provided chromatin remodeling to maintain transcription of numerous genes required for
survival, differentiation and effector function of T cells.219
T cells presence in Chronic Inflammation and Cancer
The adaptive immune system plays an important role in maintaining tissue
homeostasis. Communication between the two branches of the immune system, the
innate and the adaptive, is performed by a complex interaction of the distinctive immune
cells of each branch, their soluble factors and the environment where they are
located.198,209 In an acute inflammatory response the innate immune cells can efficiently
coordinate the adaptive immune response that leads to a rapid healing of the tissue.
However, it is known that in chronic inflammation, the innate immune system can
deregulate adaptive immune cells and rendering the overall immune response to tissue
destruction.10,137
The presence of leukocytes in the tumor microenvironment is a common feature
of many cancers. Many studies with cancer patients suggested that infiltration of T
cells, specifically CD8 phenotype, can prolong survival and improve the outcome of the
patients.222-224 However some studies showed that the excessive presence of immune
cells such as macrophages and CD4 T cells is associated with poor prognosis.225-227
CD8+ CTL have a well-described function in the tumor environment. Normally, these
cells have a specific role eliminating tumor cells. Clinical studies found that infiltration of
CD8 T cells correlated with good prognosis, although this was not a definitive
conclusion.227 This conclusion was made based on the assessment of secretion of IFNγ in the tumor environment and/or by directly performing cytotoxic tests in limited
40
number of patients.
228
CD4+ T helper lymphocytes have a more complex role in the
tumor development. Daniel et al., in a 2003 study, used K14-HPV16 carcinogenesis
model to test the possible role of CD4+ T helper cells within the inflammation
environment.229 K14-HPV mice expressed human papilloma virus 16 (HPV16)
oncogene under control of human keratin 14 (K14) promoter leading to multistage
carcinogenesis.230 Depletion of CD4+ T cells in this model delayed tumor development.
Interestingly they found that CD4+ T cells influenced the development of neoplasm
during inflammation mediated by a Staphylococcus infection.229 These findings stressed
the importance of CD4+ T cells infiltration in the inflammation process that could
enhance malignant progression.229,231 In contrast, Rao et al. observed that transfer of
pro-inflammatory CD4+ T cells to APCMin/+ transgenic mice enhanced the development
of mammary tumors.232 APCMin/+ transgenic mice are designed to develop intestinal
adenomas similar to humans that carry the APC mutation. In female APCMin/+ mice this
mutation resulted in the development of mammary tumors.232,233 The development of
tumors was attenuated by co-transfer of the inflammatory CD4+T cells with the
regulatory CD4+ T cells or by neutralization of TNFα.232 This paradoxical observation
can be explained by the deficient ability of these proinflammatory CD4+ T cells to
support humoral immunity and by the lack of help to CD8 CTL.234 All these studies
suggest that different subsets of CD4 T cells might have a distinctive role in tumor
development. CD4+ T helper cells represent a heterogeneous population of cells that
includesTh1, Th2, Th17, Th22, Tregs, and other types of cells.
41
T helper 1 (Th1) and T helper 2 (Th2) cells. The differentiation of uncommitted
CD4+ T cell to Th1 type requires exposure to IFN-γ and IL-12.235 Th1 differentiation is
regulated by the transcription factor T-box (T-bet), which also can suppress the possible
development of other types of Th cells.236 Upon differentiation, effector Th1 secrete
more IFN-γ together with other pro-inflammatory cytokines, such as TNF-α and TNF-β,
which are crucial in anti-tumor immunity as well as in the elimination of bacteria, viruses
and parasites among other pathogens.237 Activation of Th1 can induce up-regulation of
MHC class I and II molecules in APCs, which are necessary for adequate function and
durability of CD8 effector cells.238 A recent study proved that Th1 can actually acquire
MHC1/peptide complex from DCs and drive the activation and function of naïve CD8
cytotoxic T cells. They further did an adoptive transfer of CD4 Th1 cells together with
CD8 cytotoxic T cells and successfully reduced well-established EG7 tumor size in
mice.205
There is very limited information about the participation of Th1 cells in the
process of tumor formation. On the contrary, this phenotype is being described with
anti-tumor capacity and not as favoring carcinogenesis. Early studies in experimental
autoimmune encephalomyelitis (EAE), a model of autoimmunity in mice, showed IFN-γsecreted Th1 cells caused the pathology of the disease.239,240 Later, the critical role of
IL-12p40 was found, since the absence of this subunit protected the mice from various
immune disorders including EAE. Further studies showed that the deletion of IL-12p40
subunit cause a decrease in IL-23 and not IL-12.241,242 IL-23 is a cytokine secreted by
Th17, T helper cells with pro-inflammatory features.243
Th17 is an IL-17-producing
CD4+ phenotype and is going to be discussed in details below.
42
The plasticity of Th lymphocytes became focus of attention lately. For example,
some reports showed that CD4 T helper cells can secrete IFN-γ and IL-17 in inflamed
tissues.244 In Th1 polarizing conditions Th17 is capable to convert into Th1 in vivo and in
vitro. Interestingly Th1 is not capable of switching to IL-17 secreting phenotype.245,246
Adoptive transfer of polarized Th17 cells was performed in NOD mice which promote
pancreatic inflammation. However, it was shown that when these cells converted into
IFN-γ-producing Th1 it manifested in increased diabetes.247 These findings suggest
that the cytokines secreted by different CD4+ Th cells cooperate in different states of
inflammatory conditions.
As mentioned above, Th1 cells are associated with anti-tumor immunity since
they play a central role in activation of cytotoxic T cells (CTLs). However, in a colon
cancer study it was found a decreases expression of Th1 cytokines versus an elevated
expression of Th2 related cytokines.248-250 Th2 produces IL-4 and IL-10, cytokines that
are associated with immunosuppression.251 This could be explained by the presence of
polyps or adenomas, which are characterized by chronic inflammation.252 In healthy
individuals, the process of tissue restoration is characterized by the production of
suppressive cytokines, as a negative feedback mechanism in response to the release of
pro-inflammatory cytokines trying to destruct the foreign antigen. This “new”
environment allows the transformed cells to spread and metastasized. The imbalance of
Th1 and Th2 cytokines found in colorectal cancer has several mechanisms including the
synthesis of endogenous histamines and the induction of a cyclooxygenase enzyme in
immunosuppressive environments.253,254
43
Th2 differentiation essentially requires the exposure of naïve CD4+ T cells to IL-4
and IL-2. The master regulator of Th2 differentiation is GATA-binding protein 3
(GATA3).
255
GATA3 expression occurs through the up-regulation of STAT6 after the
engagement of IL-4 receptor.256 GATA3 antagonizes Th1 development.256,257 Contrary
to Th1 response, Th2 cells response is associated with humoral immunity, in which high
levels of antibodies are generated against extracellular pathogens such as helminthes
and nematodes.258 Th2 immunity is associated with the infiltration of eosinophils,
basophiles, and mast cells, which can mount a potent response against foreign
pathogens.259,260
Th2 cells can also elicit inflammation, especially in the lung.
Excessive Th2 responses are found in the mucosal tissue within the lung, which
provoke the development of atopic asthma and allergy. Cytokines IL-5, IL-4 and IL-13
secreted by Th2 cells can induce the infiltration of eosinophils in the lung.261
Interestingly, the combination of IFN-α secreted by Th1 and CD8+ cells in conjunction
with Th2 cytokines can contribute to the maintenance of the inflammation within the
lung. 262
Recently it has been proposed that Th2 type inflammation can facilitate tumor
growth and is related to poor prognosis in cancer patients.263,264. Breast and pancreatic
cancer are heavily infiltrated by Th2 cells and their signature cytokines IL-4 and IL-13.265
The studies proposed that upon activation with pro-inflammatory cytokines cancerassociated fibroblast release thymic stromal lymphopoietin (TSLPs) which act as DCs
and allowed Th2 polarization. Activation of Th2 promotes the differentiation of infiltrating
macrophages to the tumor-promoting M2 phenotype thus favoring the environment to
induce carcinogenesis.265,266
Moreover, many studies show that alteration in the
44
Th1/Th2 ratio within the tumor environment is closely related to accelerated
development of tumor and poor survival of the patient.267,268
T helper 17 (Th17) cells. Th17 CD4+ T cells were identified during the studies of
chronic inflammatory diseases such as, experimental autoimmune encephalomyelitis
(EAE) and collagen-induce arthritis (CA).269,270 Th17 cells differentiation happens when
naïve CD4 T helper cells are exposed to the pro-inflammatory cytokine IL-6 and antiinflammatory cytokine TGF-β.
271
In human and in mouse models is being shown that
IL1-β together with TNF-α can also facilitate Th17 differentiation.272 In vitro activated
DCs can secrete IL-1β and TNFα, which together with IL-6 can drive the differentiation
of Th17 but only in the presence of TGF-β.273 As the nomenclature implies, these cells
secretes cytokine IL-17 (IL-17A) but also secretes IL-17F, IL-21, IL-22, TNF-α, IL-6 and
GM-CSF among other factors.274,275
It is important to note that IL-23 have been
mistakenly implicated in differentiation of Th17 but studies confirmed that this cytokine
is required for expansion and survival of Th17 and is not necessary for initial
differentiation.276
IL-21 has more importance in the differentiation process of Th17.
This cytokine can induce the development of Th17 when IL-6 is absent or is at low
levels. It is assumed that IL-21 acts as positive feedback for maintenance the precursor
pool of Th17. Thus, under IL-6 depleted circumstances IL-21 is an important support of
Th17 responses and during development of inflammation.277 Retinoic acid receptorrelated orphan receptor gamma-t (RORγ-t) is been reported as the master regulator of
differentiation of Th17.278 IL-6 and TGF-β promote Th17 differentiation by upregulation
of RORγ-t and suppression of T-bet and GATA3 , Th1 and Th2 respective transcription
45
factors. The target genes of RORγ-t and the mechanism that induce IL-17 production
on these cells remain unclear.279 Recently, another transcription factor of the ROR
family, receptor-related orphan receptor alpha (ROR-α), was implicated in the
development of Th17. It was suggested that RORα might act cooperatively with RORγt
or as a compensation factor to Th17 since differentiation of these cells wasn’t affected
in the absence of RORα. 276
A Th17 cell function has been strongly associated with the development of
autoimmune diseases.280 The main physiological role of Th17 cells is to mount immune
defense against certain bacteria, fungi, viruses and ciliates that invade the host. These
cells have been implicated in immune defense of mucosal areas such as in the gut, skin
and lungs.281 IL-17 is a potent pro-inflammatory mediator in tissue and has a pleiotropic
effect on tissue cells and other immune cells. This cytokine can attract neutrophils by
inducing potent inflammatory cytokines and chemokines such as IL-6, IL-1-β, IL-8, TNFα, monocyte chemoattractant protein-1 (MCP-1) and can upregulate Intercellular
Adhesion Molecule (ICAM) in keratinocytes, endothelial cells, epithelial cells and
fibroblast.282 Th17 cells infiltration was observed in lupus erythromatosus, asthma,
human cancer, as well during allograft rejection and infections.283
Coccia et al.
identified synergistic activities of IL-1β and IL-23 in promoting pathogenic innate and
adaptive immune responses in the gut. Further, they were able to identified Th17 cells
derived from a T cell transfer model of colitis. Th17 cells were demonstrated to play an
important role in sustaining the inflammatory environment within the colon and
stomach.284
IL-17, produced by Th17 cells, can contribute to cancer development by
inducing angiogenesis, recruitment of myeloid cells and promoting other factors that are
46
required for cancer establishment.285,286 It was documented that the presence of IFN-γ
secreted by Th1 can deplete Th17 development.287,288 However, the coexistence of Th1
and Th17 cell populations has been reported in many autoimmune diseases. 289,290
Recently, it was demonstrated that in psoriasis inflammatory model Th1-derived IFN-γ
can activate secretion of IL-23 and IL-1β by resident APCs. This resulted in an
expansion of memory Th17 and depletion of Th1. Therefore during the development of
an inflammatory environment Th1 differentiation was diminished and was shifted
towards a Th17 type chronic inflammation by APC derived IL-1β and IL-23.285
Moreover, it has been demonstrated that Th17 cells can accelerate the tumor formation.
In a two- stage carcinogenesis study, induced by DMBA/TPA treatment, the importance
of IL17 in the development and promotion of papilloma in the skin was shown. This
effect occurred via activation of STAT3 by IL-6 which was induced by IL-17.291 However,
many other studies showed anti-tumor activity of Th17. Muranski et al. showed that
polarized IL-17 secreting CD4+ cells have a better anti-tumor function than Th1 cells.
They noted that Th17 cells were able to produce IFN-γ which is known to have a potent
anti-tumor function.292 This also contributed to the concept that different types of Th
cells exhibit functional plasticity.278,293 In another study, it was demonstrated that Th17
were able to stimulate tumor rejection through activation of CD8+ T cells. Ovalbumin
(OVA)-specific Th17 cells acquired peptide/MHC1 and secreted IL-2, which activated
anti-tumor functions of CD8+ CTL but not thru IL-17. This study provides the base idea
to design preventive Th17-based anti-tumor therapy. 294
Other findings strengthen the idea of a possible plasticity or inter-phenotype
change within the T helper cells populations. In another study, it was shown that human
47
Th17 can secrete IL-17 and IFN-γ and express ROR-γt and T-bet (Th17 and Th1,
respectively) within the gut of Crohn’s disease (CD) patients.
Expression of IFN-γ by
differentiated Th17 was attributed to the presence of IL-12.295 These findings could be
expected since IL-23 is the cytokine necessary for Th17 maintenance and belongs to
the IL-12 family of cytokines.296 Further this same study revealed that the chemokine
receptor CCR6 was expressed in Th17 cells.295 CCR6 was up-regulated in memory
CD4+ T cells and B cells but not in naïve CD4 + T cells or Th2 cells.297,298 Its ligand,
CCL20, have being shown to selectively attract memory T cells.298
The selective
expression of CCR6 in Th17 and Th1/Th17 population suggested that these cells may
remain activated long time after its activation and therefore, could be attracted to the
site of inflammation.
Th17 cells can also acquire characteristics of T regulatory cells (Tregs). Inverse
correlation
of
the
number
of
Th17
and
Tregs
was
found
in
tumor
microenvironment.200,299 This relationship became evident when it was discovered that
these cells shared requirement of TGF-β in the differentiation from naïve CD4+ T cells
into anti-inflammatory Tregs and into pro-inflammatory Th17 phenotype.300
As
discussed above the development of Th17 apart of TGFβ, strictly requires the presence
of a pro-inflammatory cytokines, like IL-6, derived from activated DCs, or other cells
present in the inflammatory environment. Therefore, in an environment deprived of proinflammatory cytokines most likely naïve CD4+ T cells have potential to differentiate into
Tregs.301 Differentiation of functional Tregs requires the expression of Forkhead Box
P3 (FoxP3) transcription factor which is necessary for its development and
maintenance.302 Interestingly, uncommitted T cells begin to express both ROR-γt and
48
FoxP3 in the presence of TGFβ alone but failed to express IL-17. Eventually it was
noted that ROR-γt expression was diminishing and Tregs development was more
apparent. Likewise, a transient co-expression of ROR-γt and FoxP3 was seen early
differentiation of Th17, in which FoxP3 was gradually down-regulated.303,304 Moreover,
in a human study it was shown that different subset of myeloid cells isolated from
peripheral blood can induce differentiation of Tregs or Th17 in a TGF-β-dependent
fashion. MDSCs, defined with the surface markers CD14+HLA-DRlow/- , and monocytes,
with surface markers CD14+HLA-DR+, isolated from peripheral blood were able to
differentiate Tregs and Th17 respectively.305 These two myeloid cell populations
express TGF-β, which is an essential requirement for Tregs and Th17 differentiation.
These particular MDSCs did not express pro-inflammatory cytokines, like IL-6,
necessary for Th17 generation and therefore contributed to FoxP3 up-regulation and
Tregs development. On the other hand monocytes could secrete the inflammatory
cytokines and induce differentiation of Th17 by upregulating of ROR-c. These findings
suggest that the environmental factors can determine the final effector phenotype of T
cell population thus regulating the outcome of the disease. More importantly, this work
further demonstrated that the MDSCs were able to differentiate Tregs from Th17 cells
via a retinoic acid (RA)-dependant mechanism. It has been shown that RA, a metabolite
of vitamin A, can induce Tregs’ differentiation by suppressing IL-6 induction of Th17
cells. Upon addition of RA agonists to the co-culture of differentiated Th17 and MDSC,
MDSCs enhanced the conversion of Th17 into FoxP3-expressing Tregs cells. Further,
blocking the retinoic acid receptor or TGFβ receptor demonstrated a reduction of the
conversion of Th17 into T regulatory cells.305 This is an important observation because
49
it can partially explain the mechanisms underlying the pro-inflammatory versus
suppressive immunity within the tumor environment.
T regulatory cells (Tregs). Tregs are developmentally and functionally different
T cell subpopulation that contribute to self-tolerance.306 Although these cells are being
identified by expression of CD25 surface marker this marker doesn’t discriminate
among other effector T cells and suppressive Tregs. FoxP3 has been identified as the
principal transcription factor that allows the development of suppressive Tregs.307,308
Two subpopulations of CD4+ Tregs were identified: thymus-derived natural occurring
CD4+CD25+FoxP3+ Tregs (nTregs), and those that can be induced ex-vivo from CD25precursors in peripheral lymphoid organs (iTregs). These two subpopulations can be
found within the same environment and contribute to immune suppression.309,310 The
main function of these cells is to execute its suppressive activity against CD4+CD25- T
cells, CD8+ T cells, B cells and Natural Killer (NK) cells in a cell to cell contact and/or
secreting immunosuppressive cytokines such as IL-10 and TGF-β. This suppressive
activity allows down-regulation of excessive immune responses and maintains T cell
tolerance to self-antigens.311,312 Expansion of Tregs in cancer favors tumor development
and progression through the inhibition of anti-cancer responses. As mentioned above,
TGF-β is the cytokine necessary for Tregs cell differentiation from naïve CD4+ T cells.
Generally, TGF-β has been identified as an immunosuppressive cytokines but this
cytokine has pleiotropic functions in T cell responses.313 TGF-β supports the
maintenance of FoxP3 expression, the suppressive function and homeostasis in
peripheral Tregs.314 TGF-β also has been implicated in the role of retinoic acid (RA) in
50
the Tregs development.
RA can interfere with inhibitory cytokines that prevent
development of Tregs and further can enhance expression of FoxP3 transcription
factor.315,316
The increased presence of Tregs in the tumor environment often correlates with
poor prognosis.
Moreover, detection of a high frequency of Tregs can prevent
successful anti-tumor therapies in cancer patients.317 Animal model experiments also
corroborate the participation of Tregs in tumor progression. Depletion of these cells is
associated with tumor rejection, which is probably can be explained by a change in
cytokine availability. Tumor cells and tumor environment are capable of inducing DC to
secrete TGF-β thus promoting Tregs’ differentiation from peripheral naïve CD4+
cells.137,318 The expression of chemokines and chemokine receptors can contribute of
recruitment of Tregs to the tumor environment. Activated Tregs can express CCR4 and
therefore can be recruited to the tumor environment by tumor cells and macrophages
that express CCL22, the ligand of CCR4.319 The hypoxic environment found in ovarian
tumors secretes CCL28 which in turn attract Tregs via the engagement of CCL28 and
its receptor CCR10 that is present in these regulatory cells.320 Once the Tregs reach the
tumor environment, they contribute to immune attack suppression and angiogenesis by
in the tumor environment, Tregs contribute to immune suppression by secreting TGFβ,
IL-10 and IL-35.319,321
Chemokines and Inflammation
It has been demonstrated that chemokines can promote carcinogenesis by
setting the condition for the onset and progression of inflammation.322 Although
51
chemokines can have biological purposes, which includes regulation of hematopoiesis,
fibrosis and angiogenesis, these chemoattractants can operate as a signal to guide
cellular migration which coordinates leukocyte recruitment in physiological and
pathological conditions.323,324 Chemokines can be divided into two groups depending
on
their function:
“inflammatory”
chemokines
and
“homeostatic”
chemokines.
Inflammatory chemokines mediate trafficking of leukocytes to the inflammations site and
help develop strong immune responses. Homeostatic chemokines are constitutively
expressed, involved in lymphocyte and DC trafficking, and can recruits cells to the
hematopoietic organs.325 Inflammatory chemokines mediate the interaction among
leukocytes, lymphocytes, tumor cells, stromal cells with each other resulting in tumor
growth and dissemination.326,327 The first modification of the resident cells within the
chronic environment leads to the secretion of non-specific pro-inflammatory cytokines,
such as IL-1α/β, IL-6, TNF-α and IFN-γ and these molecules subsequently can induce
the secretion of other pro-inflammatory chemokines. These chemokines and cytokines
can act in a paracrine and autocrine fashion helping to magnify of inflammatory
responses.328 All these events contribute to the persistence of inflammation which
promotes the conversion of normal cells to pre-neoplastic cells.329
Chemokines are small peptides (8-17 kD) secreted by cells to attract other cells
to the microenvironment. Chemokines comprise the largest family of cytokines. Is been
classified into CC, CXC, C, and CX3C depending on their relative positions of
conserved cysteine residues in the mature sequence of the protein. Chemokines binds
to its receptor, a family of 7 transmembrane domain G-protein coupled proteins. There
are 4 families of chemokines receptors: CC, CXC, CX3C and X3.322 Two steps have
52
been proposed to describe the action and activation of chemokines and its receptors.
The first step is the recognition of the chemokine with its specific receptor. The second
step
relies
on
the
activated
receptor.330
Chemokine
receptor
undergoes
a
conformational change upon chemokine binding, which causes an exchange of GDP to
GTP in the α-unit of the G protein. This results in the activation of a downstream
signaling cascade, which in part leads to the expression of integrins allowing attachment
of the leukocytes to the endothelial surface and subsequent extravasion.331
The
activation of the receptor and up-regulation of integrin molecules on the cell surface are
needed for complete activation of leukocytes, enhancement of phagocytosis,
superoxide production, granules release and microbicidal activity. 323
It is important to
note that many chemokines can bind to more than one receptor with high affinity, and
similarly, that some chemokine receptors don’t discriminate with different chemokines.
This promiscuity allows for the development of a large array of coordinated
responses.332 Recent studies have identified chemokines and chemokine receptors that
elicit and sustain inflammatory events in the environment that could evolve into
cancer.322
Consequences of the engagement of chemokines and its respective
receptors are activation and/or inhibition of pathways within targeted cells that could
accelerate the process of carcinogenesis.333 Mononuclear cells can be attracted by
chemokines to the inflammation site.334 Anti-inflammatory cytokines, such as IL-10, can
induce up-regulation of pro-inflammatory chemokine receptors CCR2, CCR5 and CCR1
in monocytes.335 On the contrary, the same chemokines receptors were down-regulated
in the presence of a pro-inflammatory environment.336 This effect might provide an
inhibitory signal to prevent excessive macrophage recruitment at the sites of
53
inflammation and tissue damage.337 The involvement of chemokines in the recruitment
of immature myeloid cells to the tumor environment depends on the phenotype of the
myeloid cells and the tumor model. From the four known families of chemokines, CC
family is the predominant family in the infiltration by TAMs and T cells.338. In early
stages of cancer development, IMCs expressing CCR1 are recruited by CCL9, which is
produced in the tumor epithelium.339 More specifically, it has been demonstrated that MMDSC are recruited to melanoma tumor environment through interaction of CCL2 with
its receptors CCR2, CCR4 and CCR5.340,341 Other groups reported the participation of
different chemokines in the migration of MDSC to the tumor site. In ovarian and gastric
cancer it has been shown the participation of CXCL12 and CXCL8 (also known as IL8).342,343 Animal models also showed the chemokine-induced MDSC migration towards
the tumors. Sawanobori et al. demonstrated that M-MDSCs were attracted to the tumor
site by CCL2/CCR2 interaction; meanwhile, PMN-MDSCs were attracted by CXCR2
ligands secreted by tumor cells.340
These studies supported the active and important
role of chemokines in IMC/MDSC destiny and their contribution to cancer
progression.128,340,344
Chemokines can expand its influence to T cells lineages.345
Tumor infiltrated CD8+ cells that express CCR7 are positive prognosis markers in colon
cancer patients. Once CCR7 is engaged to its ligands, CCL21 or CCL19 can mobilize
CD8+ cells to lymph nodes or to the tumor environment and executes its cytotoxic
response.346 In a study of breast cancer, Curiel et al. have shown that Tregs expressing
CCR4 are attracted to the tumor environment by CCL22 secreted by tumor associated
macrophages and tumor cells. 347
54
It is clear that the cells and molecular components that induce chronic
inflammation influence tumor development. Knowledge and understanding of these
complicated processes driving the inflammation and tumor development could lead to
the development of new therapies to counteract the malignant progression.
Macrophage Inflammation Protein-1 beta (MIP-1β) / CCL4. The first
characterization of this chemoattractant protein was made in the late 80’s when murine
macrophages were stimulated with LPS which induced the secretion of multiple factors
including a new and unidentified monokine. This heparin-binding protein attracted PMN
when was injected to mice and rabbits, where it could act as an endogenous pyrogen
and induce granulocyte-macrophage colony formation.348 In in-vitro studies showed that
CCL4 attract PMN and induce superoxide production in human PMN
348,349
Because of
these inflammatory properties, this protein was named as macrophage inflammatory
protein (MIP).349 Further, SDS-PAGE assay was able to identified two distinct protein
doublets that were highly related, MIP-1α and MIP-1β with 68% homology.350 MIP-1β or
chemokine (CC-motiff) ligand-4 (CCL4) is the member of the MIP family that include
MIP-1α (CCL3), MIP-1δ (CCL9/10) and MIP-1γ (CCL15).351
These are 8-10 KDa
proteins.322 In mice and humans, CCL4 is encoded by the genes consisting of three
exons and two introns positioned in chromosome 17.351 This chemokine is synthesized
as an immature precursor of 92 aminoacids. After subsequent cleavage in hydrophobic
specific aminoacids by peptidases, the mature protein consists of 69-70 aminoacids.
350
Generally, CCL4 and CCL3 production can be induced by different pro-inflammatory
cytokines/agents, such as LPS, IFN-γ, IL-1β, TNF-α, viruses, etc. Anti-inflammatory
55
cytokines such as IL-4 and IL-10 can down-regulate its expression. Upon stimulation
with LPS or IL-7 human monocytes can secrete significant amounts of CCL4. In addition
lymphocytes are capable of secreting CCL4 upon antigen binding.352 CCL4, secreted
by PMN in the skin, is found to be an important mediator of macrophages migration in a
murine model of cutaneous granuloma formation.353 In parasite infection, CCL4 can
induce the secretion of IL-12 by resident DCs, which contribute to Th1 infiltration which
facilitate the clearance of the worm by cytokine effect.354
Recently, in a model of
tumorigenesis of prostate cancer, it was demonstrated that CCL4 participates as a
mediator of macrophages to promote malignant differentiation of immortalized prostate
epithelial cells.355
The recognized receptor for CCL4 is CCR5, although in HIV models a modified
version of CCL4 can signal through CCR1 and CCR2b.350,356 This receptor is expressed
mainly in subpopulation of lymphocytes and in monocytes/macrophages.350,357
Signaling through any of these receptors induces the internal rearrangement of the G
proteins.
This new conformation of G proteins results in the activation of
phosphoinositide 3-kinase (PI3K) pathway and phospholipase C signaling pathway
which permits the influx of Ca2+ and activation of protein kinase C (PKC).351
Soon
after its discovery, CCR5 is recognized as co-receptor that facilitates the entry of HIV-1
virus in CD4 T helper cells. The gp120 glycoprotein of the HIV binds to CD4 protein in
the target cells, which cause a conformational change in gp120. This rearrangement
allows the entry of the virus.358 CCR5 has been implicated as a possible mechanism of
cancer progression.
Tan et al. demonstrated that Tregs participated in early
development of pancreatic cancer via CCR5 expression.
56
Administration of small
molecules CCR5 antagonist was able to reduce infiltration of Tregs to the tumor site and
slow down the tumor growth in mice.359
For many years different chemokines have been implicated in the migration of
leukocytes to the site of injury and infections.360 Therefore, it is conceivable that MIPs
family can help orchestrate pathogenesis in many inflammatory conditions and
diseases, including cancer.351,360 However, there is limited data demonstrating that
cancer cells can also secrete MIPs, particularly CCL4. In an in-vitro experiment, Burger
et al. demonstrated that Chronic Lymphocytic Leukemia (CLL) cells co-culture with
monocyte-derived nurse-like cells (NLC) express high amounts of CCL3 and CCL4.
The induction of CCL3 and CCL4 was a result of the activation of CCL B cell receptor in
response to antigen presented by NLC.
Secretion of CCL4 and CCL3 support
interaction of CCL with T cells and might explain maintenance of tumor
microenvironment and CCL diffusion to other organs.361 Erreni et al. have identified
highly expressed chemokines and chemokines receptors in tumor tissues from human
colorectal cancer. Among the chemokines they found that CCL4 andCCL3, were highly
expressed in the tumor samples compared to normal tissue. It is known that these
chemokine are ligands for CCR1 and CCR5-expressing monocytes/macrophages but
they couldn’t decipher if these infiltrated myeloid cells are pro-tumor or anti-tumor.
Moreover, they couldn’t associate the, the up-regulation of these chemokines with the
stage of the tumor.362
57
Myeloid cells induce T cell migration towards the inflammatory site
It is well documented that TAMs and MDSC influence tumor development.363,364
The migration of these myeloid cells into the neoplastic tissue can aid in the stroma
remodeling, angiogenesis, and the influx of lymphocytes that contributes to cancer
formation.
Besides the well-studied suppression mechanisms against CD8+ CTLs
these myeloid cell populations secrete other inflammatory factors, which contribute to
the persistence the inflammation.137 It has been reported that pro-inflammatory
cytokines, such as IL-6 and IL-1β, not only contribute to myeloid cells recruitment and
proliferation but also can help the infiltration of lymphocytes into the tumor
microenvironment.365 As discussed above, one function of pro-inflammatory cytokine
IL-6 is to promote the differentiation of naïve CD4 lymphocytes into the proinflammatory phenotype, Th17, in the presence of TGFβ.271 Th17 can counteract the
tumor suppressive IFNγ-secreting Th1 in the tumor environment.366 Several studies
have shown that among TILs population, Th17 found at high percentage in different
types of cancer.299,367,368 Upon arrival to the neoplastic tissue effector Th17 can secrete
pro-inflammatory cytokines, such as IL-17, IL-1β, IL-23, which generally favor tumor
growth but also can limit tissue damage depending on the site of the tissue site .369
Chemokines are the primary molecules that drive the infiltration of myeloid cells
into the tumor environment.98,344. Further the myeloid cells that are present in the tumor
environment can also secrete chemoattractants to induce the infiltration of
lymphocytes.370 CCL17, CCL22,
CCL24 can support the immunosuppressive
environment by inducing migration of Tregs to the inflammatory site.139,371 Further, IL-4
secreted by Th2 can attenuate CD8+ T cells cytotoxic immunity and induce the
58
development
of
immunosuppressive
macrophages.136,226
Within
the
tumor
microenvironment, TGF-β secretion is not limited to macrophages only. MDSCs are
also able to produce this cytokine which contribute to their immunosuppressive
function.372 In addition, TGF-β produced by MDSCs can favor the differentiation and
infiltration of naïve CD4+ T cells to T regulatory cells. Within the emerging cancerous
tissue, chemokine productions by MDSCs and TAMs also have been implicated as
immunosuppression mechanisms within the tumor site. In the Lewis Lung Carcinoma
(LLC) model, CCL22 secreted by these myeloid cells and not by tumor cells can
promote the infiltration of CCR4 expressing Tregs which collaborate into promoting the
suppressive environment thus favoring cancer development. Interestingly, in this model
CCL22 was produced by MDSCs and not by tumor cells.
373
Chemokines also play an important role in the tumor development. Chronic
inflammation is characterized by the persistent infiltration of leukocytes. Oo et al.
reported that during chronic liver inflammation DCs from the affected area secretes the
CCR4 ligands, CCL22 and CCL7, which recruits Tregs thus showing to immune
suppression within the environment.374 In another study, Yurchenko et al. reported that
infection with parasite Leishmani become persistent with the infiltration of Tregs in
dermal sites. Upon infection with the parasite CCR5 ligands CCL3, CCL4 and CCL5 are
secreted possibly by DC, infected macrophages, or effector T cells which enable CCR5+
Tregs to migrate to the site of infection. Further, they found that in CCR5KO mice were
resistant to the parasite infection which correlates with Th1 responses seen on the site
of infection.375 Himmel et al. have shown that it was possible that activated human
Tregs could secrete chemokines, specifically CXCL8, to improve its immunosuppressive
59
activity. They also showed that other chemokines, such as CCL3, CCL4, were secreted
upon stimulation with CD3 and CD28 antibodies and that these chemokine-secreted
Tregs were able to recruit PMN.376
It is important to note that due to the nature of the process of inflammation and
eventual carcinogenesis the expression of chemokines within this environment attracted
mainly effector or memory T cells.377 These activated lymphocytes, mainly CD4+ T
cells, have high expression of chemokine receptors and respond to respective ligands
by migrating to the inflammatory site.378
All these studies strongly suggest the importance of chemokines and chemokine
receptors within the inflammatory environment and its contribution to the infiltration of
leukocytes. Understanding of the mechanisms of their recruitment may help to develop
novel therapies that can inhibit the possible development of cancer.
Tumor cells need Inflammation for survival and metastasis
Cancer cells can elicit the inflammation to support the proliferation and survival of
malignant cells. The stimulus within chronic inflammation can promote cancer survival,
new vessel formation, and metastases. The formation of metastasis can be supported
by inflammatory environment, which can suppress immune response and interfere with
immuno- and chemotherapeutic drugs.379
380
One of the most important elements of
cancer metastasis is the ability to move to distant organs and/or tissues through the
blood circulation, lymphatic vessels or serosa surfaces.381 Recently, it was proposed
that the tumor cells within a tissue are not a uniform population. Tumors contain
population of cells that may contribute to tumor aggressiveness. Cancer Stem Cells
60
(CSCs) are tumor cells able to derive new tumor cells when implanted in vivo. Another
important trait of CSC is the ability to evade apoptosis, distinct mobility, and
invasiveness.382 It was suggested that metastatic cancer cells undergoing epithelialmesenchymal transition (EMT), enabling them to invade distant tissues by reducing
signatures adhesion molecules, such as E-cadherin. EMT in cancer cells is supported
by
the
tumor
microenvironment
characterized
by
inflammatory
cells
and
molecules.383,384
The inflammatory cells provide additional factors that can potentiate the tumor
cells to become invasive. For example, TAMs (the cells that predominate in the tumor
environment) can secrete a wide variety of growth factors and cytokines that aid in the
motility, growth and invasiveness of tumor cells.
In addition, TAMs can secrete
proteinases that degrade the basal membrane creating a channel for tumor invasion.
TNF-α secreted by the TAMs dramatically enhances the invasiveness of tumor.379,385
Further, metalloproteinases (MMP) secreted by TAMs can also contribute in tumor
progression. In a lung cancer model, the functional importance of MMP-9 secreted in
the neoplastic tissue was confirmed. The MMP-9 found in the lung was prior localized in
distant tumor prior metastasis.
A decrease in lung metastasis was found in MMP-9
deficient host suggesting the functional importance in the pre-metastasis sites. 386
Recent studies have shown a close correlation between the level of MDSCs and
cancer stage, metastatic tumor burden and chemotherapy responses. Several studies
suggest two hypothesis in which MDSCs participate in tumor invasion and metastasis.
387
The first one is that MDSCs can secrete factors, such as MMP and chemokines that
can prepare the environment for tumor progression and metastasis.387-389 The second
61
one is that tumor cells can apparently can usurp or take over MDSCs machinery and
use it for self-maintenance enabling them to metastasize.390,391 Further, S100A8 and
S100A9, the proteins that are up-regulated in MDSCs, have been shown to contribute to
tumor invasiveness.392 Within the established tumor environment these proteins
promote the phosphorylation of MAPK and activation of NF-κB which allows migration of
the neoplastic clone.393,394 Addition of S100A8/A9 into B16 melanoma cells in which
activates matrix metalloproteinases that is associated with tumor metastasis.395
Moreover, in early 2000s, Taguchi et al. reported that the blocking RAGE, the receptor
for S100A9 protein, can decrease metastasis in vivo, as well cell growth and
migration.396
High levels of these proteins are correlated to poor prognosis in cancer patients.
397
The fusion of bone marrow derived cells (BMDC) and tumor cells is another
described phenomenon that contributes in tumor metastasis. These hybrids have been
detected in several animal models and in human cancer. Molecular studies suggest that
these hybrids up-regulate genes associate to metastatic cancer cells and genes
associated with migratory macrophages and other BMDC.
391
CXCR4 is the chemokine receptor strongly associated in metastatic cancer.322,398
The chemokine ligand that binds to CXCR4 is CXCL12 also known as stromal cell
derived factor 1 (SDF1).399 This chemokine/chemokine receptor complex is related to
trafficking of HSC andvascularization, as well as to heart and brain development.
CXCL12 is considered a hematopoietic chemokine and its principal function is to
regulate hematopoietic cell trafficking and formation of secondary lymphoid organs.
However, CXCL12 is also associated with tissue injuries such as heart infarction, liver
62
toxicity, and adverse consequences chemotherapy treatment.400 There is abundant
evidence suggesting that chemokines can influence the traveling of primary tumors to a
secondary lymphoid location by up-regulating genes related to cell survival, cell
adhesion and other genes transcription that coordinate trafficking of cells.401 The
overexpression of CXCR4 has been observed in more than 20 different types of cancer,
including melanoma, ovarian, glioma and pancreatic.402 Interestingly, CXCR4 is highly
associated with CSC and has been implicated in survival and metastasis of
gliobastoma tumor cells and pancreas tumor cells.98 CXCR4 is expressed in a variety of
cells BM derived cells including, early T and B lymphocytes, CD34+ cells, monocytes,
granulocytes and endothelial progenitor cells among others.
These CXCR4+ cells
exhibit pro-angiogenic properties which include infiltration of CXCL12 secreting cells for
neovascularization and support of tumor growth.400,403
As we discussed previously, early stages of cancer, characterized by
inflammation, have many different cells that interact with each other and confabulate to
create a neoplastic environment. It has become well known that myeloid cells,
specifically MDSCs, participate in chronic inflammation that leads to tumor formation as
well as in other inflammatory diseases. This project intends to identify the specific role
of MDSC in tumor development and possible mechanism of their involvement.
Hypothesis
Immature myeloid cells accumulated during chronic inflammation and promote
tumor development.
63
Aim #1. To investigate the role of immature myeloid cells and MDSC in skin
tumor development.
Aim #2. To determine the role of MDSC and immature myeloid cells in the
development of lung cancer.
64
CHAPTER 2. IMMATURE MYELOID CELLS FOSTER
TUMOR DEVELOPMENT VIA RECRUITMENT OF CD4+ T CELLS
Rationale
There is now ample evidence of abnormalities in the myeloid compartment in
cancer, which manifests in inhibition of differentiation of dendritic cells (DC), polarization
of macrophages (MΦ) towards M2 functional state, and dramatic expansion of myeloidderived suppressor cells (MDSC).136 MDSCs represent a heterogeneous population of
pathologically activated myeloid cells that includes precursors of neutrophils, MΦ, DCs
cells, and cells on earlier stages of myeloid cell differentiation.147,151 In mice, these cells
are defined as Gr-1+CD11b+ cells with the recognition of polymorphonuclear (PMNMDSC) and mononuclear (M-MDSC) subsets based on the expression of Ly6C and
Ly6G markers.156,178,180,404 PMN-MDSC largely consists of pathologically activated
immature neutrophils, whereas M-MDSC are pathologically activated monocytes.159
MDSCs are characterized by a potent immune suppressive activity and the ability to
promote tumor angiogenesis, tumor cell invasion, and metastases.151,405 In tumor-free
mice, cells with the same phenotype represent immature myeloid cells (IMC) as well as
neutrophils and monocytes lacking immune suppressive activity. Expansion of MDSC is
considered a consequence of tumor progression. However, in recent years, it has
become clear that the cells with phenotypes and functions attributed to MDSC are
readily detectable at different conditions associated with chronic inflammation, sepsis,
65
transplantation, etc. not directly linked to cancer.406
Inflammation is developed in response to foreign agents or injuries and is an
important part of the healing process. However, if inflammation is not resolved and
becomes chronic, it may become an important contributing factor in the development of
cancer.5
Myeloid cells together with lymphocytes are the main components of
inflammation. The exact nature of myeloid cells that trigger initial tumor development as
well as the nature of their interaction with lymphocytes is not clear. Since cells with
MDSC phenotype are frequently associated with chronic inflammation, we were
interested in establishing their involvement in initial steps of tumor development. This
may have potential therapeutic implication since strategies to target MDSC are currently
being developed for clinical purposes.136
In order to formally address this question, one needs to set up a condition where
immature myeloid cells (IMC) accumulate in tissues without confounding effects of
various pathologic factors. To achieve this goal we used experimental models with
regulated expression of S100A9 protein, the member of S100 family of Ca 2+ binding
proteins.167 Expression of S100A9 together with its dimerization partner S100A8 is
found predominantly in cells of the myeloid lineage. Promyelocytes differentiating to
myelocytes/granulocytes increase expression of S100A9, whereas differentiation of MΦ
and DCs is associated with loss of S100A9/A8 expression.
Cells of the lymphocytic
lineage do not express these proteins. We and others have previously found that
accumulation of MDSC and inhibition of DC differentiation in cancer were closely
correlated with upregulation of S100A8/A9.167 The expansion of MDSC was significantly
reduced in S100A9 deficient tumor-bearing mice and mice treated with complete
66
Freund’s adjuvant (CFA).167 Furthermore, this was consistent with more recent report
that DCs derived from S100A9 deficient mice induced stronger response of allogeneic T
cells. On the other hand, overexpression of S100A9 in transgenic mice resulted in
inhibited DCs and MΦ differentiation.167 These results suggested that manipulation of
S100A9 in myeloid cells could facilitate the accumulation of IMC and thus open the
opportunity to directly test the role of these cells in tumor development.
Results
The role of immature myeloid cells in tumor development.
Tumor
development was studied in two models of skin carcinogenesis. Tg.AC mice on FVB/N
background express the mutant v-h-ras transgene under the zeta-globin promoter.
When treated topically with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate
(TPA), Tg.AC mice develop multiple papillomas, some of which progress to a malignant
stage.407 In C57BL/6 mice, skin tumor formation was induced by a single application of
the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) followed by 12-week topical
treatment with TPA. To test the role of IMC in tumor development we used transgenic
mice expressing the S100A9 protein in hematopoetic cells (S100A9Tg mice).167 These
mice on FVB/N background had no detectable abnormalities during the first 4-5 months
of life. Compared to wild-type (WT) mice, S100A9Tg mice expressed higher amounts of
S100A9 protein in bone marrow (BM), spleen, lymph nodes (LN), and lung, but not in
skin or liver (Figure 4A). S100A9 could be induced in keratinocytes under inflammatory
conditions. However, topical treatment of WT mice with TPA for 4 weeks did not result
67
in detectable levels of S100A9 in skin. In contrast, the same treatment of S100A9Tg
mice caused a substantial increase in the amount of the protein (Figure 4B).
B
A
Figure 4. Expression of S100A9 protein in different organs of WT and S100A9tg
mice: (A) Basal amount of S100A9 protein expressed in different organs of WT and
S100A9tg mice. (B) Expression of S100A9 protein on skin of S100A9tg and WT mice
after treating with TPA or vehicle (acetone). Each lane represents one mouse. Shown
is typical Western Blot with 30ug protein total.
We assessed the presence of myeloid cells in skin by immunohistochemistry.
While treatment of WT mice with TPA resulted in a small increase in the number of Gr1+ cells, in S100A9Tg mice these cells increased ~3-fold (p<0.01) (Figure 5A, B).
Evaluation of cells derived from skin sample digests by flow cytometry revealed that all
the Gr-1+ cells also expressed CD11b. The proportion of Ly6ChiLy6G- monocytic cells
among CD11b+ cells in TPA-treated skin was the same in WT and S100A9Tg mice,
whereas the proportion of Ly6CloLy6G+ granulocytic cells was dramatically higher in
S100A9Tg than in WT mice (Figure 5C). No differences in the numbers of F4/80+ MΦ
between WT and S100A9Tg skin were found regardless of treatment (Figure 5D),
whereas the number of CD11c+ cells was significantly lower in both acetone and TPA
treated S100A9Tg skin than in WT skin (Figure 5E).
68
.
A
WT-TPA
Gr1
B
S100A9tg-TPA
**
40
**
30
c e lls /m m
2
*
20
*
10
A
c
-T
P
-A
g
T
T
9
S
1
S
0
1
0
0
A
0
W
A
T
9
-
W
T
T
g
P
-A
A
c
0
WT
C
WT
S100A9Tg
*
30
S100A9tg
% of CD11b+ cells
25
20
15
10
5
D
E
F480
8
2
c e lls /m m
4
hi
Ly
6C
Ly
6C
hi
6G
-
6G
+
6G
lo
6C
CD11c
15
10
c e lls /m m
2
6
Ly
Ly
6C
lo
6G
+
0
5
*
**
2
0
P
-T
tg
ce
A
9
-A
S
1
S
S
0
0
1
0
A
0
9
T
W
A
0
0
1
A
e
n
to
P
T
W
-A
-T
to
P
-T
tg
9
-A
9
A
S
1
0
0
W
ce
A
e
n
to
ce
-T
T
W
T
-A
ce
to
P
n
A
e
n
A
e
0
Figure 5. The presence of myeloid cells in the skin of WT and S100A9tg mice: (A)
Representative picture of Gr1 staining in immunohistochemistry assay. Scale
bar=100um. (B) Graph of Gr1+ cells per mm2 of skin tissue. Each group combined 4
mice. Mean and SD is shown (*p<0.05, **p<0.01). (C) Phenotypic characterization of
myeloid cells on TPA-treated skin. Flow cytometry plots are shown (left), numbers
represents gated cells. Bar graphs represent the distribution of cells (right), *p<0.05 (D)
Graph of F480+ and (E) CD11c+ cells of TPA-treated mice. Each group included 4
mice. Mean and SD is shown, (*p<0.05, **p<0.01)
69
We asked whether dramatic increase in the presence of granulocytic IMC in the
skin of S100A9Tg mice was the result of expansion of these cells. There was a slight
increase in the presence of IMC in BM of TPA treated S100A9Tg versus WT mice but
the ratio between granulocytic and monocytic subsets of IMC was the same (Figure 6A,
B). The presence of MΦ in BM of S100A9Tg mice was slightly lower than in WT mice
(Figure 6C). These data indicate that consistent with previous observation 167
overexpression of S100A9 in myeloid progenitors did not cause expansion of myeloid
cell population but rather inhibited their differentiation. Topical application of TPA in
S100A9Tg mice caused a substantial accumulation of IMC with granulocytic phenotype,
whereas the presence of MΦ and DCs was either unchanged or decreased.
To study skin carcinogenesis, the S100A9Tg mice were crossed with
homozygous Tg.AC mice, and hemizygous monotransgenic (Tg.AC) and bitransgenic
(S100A9:Tg.AC) littermates were topically treated twice weekly for 6 weeks with the
tumor promoter TPA. Papillomas developed much faster and at significantly (p<0.001)
higher numbers in bitransgenic mice expressing S100A9Tg than in monotransgenic
mice (Figure 7A). To clarify the role of infiltrating BM derived cells in increased
tumorigenesis in S100A9Tg mice, lethally irradiated Tg.AC mice were reconstituted with
BM from WT or S100A9Tg mice and papilloma formation similarly evaluated after TPA
treatment. Recipients of S100ATg BM developed significantly (p<0.05) more papillomas
than recipients of BM from WT mice (Figure 7B) demonstrating that upregulation of
S100A9 in BM-derived hematopoietic cells was responsible for increased tumor
formation. To confirm specific role of IMC in tumor development, Gr-1+ cells were
depleted in Tg.AC mice with Gr-1 antibody during the first three weeks of TPA
70
treatment. This treatment significantly (p<0.05) reduced papilloma formation (Figure
7C).
To better ascertain the possible role of IMC in tumor development we also used
mice with targeted deletion of S100A9 (S100A9KO mice, C57BL/6 background).194 In
contrast to FVB/N mice, untreated WT C57BL/6 mice had traces of S100A9 protein in
the skin, which was increased after 4 weeks of TPA treatment (Figure 8A). S100A9KO
mice had no detectable S100A9 protein in the skin (Figure 8A). Treatment of C57BL/6
mice with TPA greatly increased the number of Gr-1+ myeloid cells in the skin, whereas
in S100A9KO mice, this increase was very modest and lower than in WT mice (Figure.
8B). In contrast to S100A9Tg mice, S100A9KO mice developed significantly (p<0.01)
fewer skin tumors than their WT C57BL/6 counterparts after treatment with both DMBA
and TPA (Figure 8C). Mice reconstituted with BM from S100A9KO mice had
significantly (p<0.01) less number of lesions than mice reconstituted with WT BM
(Figure 8D), confirming the critical role of BM derived cells in modulating skin
tumorigenesis. Collectively, these data demonstrate that development of skin tumors
directly depended on the accumulation of granulocytic IMC in the skin. Next we
investigated the mechanism(s) by which IMC mediated tumor promotion.
Accumulation of IMC in skin did not result in immune suppression. We
tested the hypothesis that IMC accumulated in TPA-treated S100A9Tg skin had
immune suppressive activity and thus could be classified as MDSC. Since isolation of
71
A
B
C
Gr1+CD11b+ cells
Ly6G+/Ly6C+ cells
F480+cells
Figure 6. Myeloid cells accumulation in the bone marrow after TPA treatment of
mice skin: Distribution (left) and absolute number (right) of (A) Gr1+CD11b+ cell (B)
Ly6C/Ly6G ratio within the population of CD11b+ cells and (C) F480+CD11b+ cells in
bone marrow of TPA treated mice. Each group included 4 mice. Mean and SD are
shown. * p<0.05 between groups
72
A
B
C
Figure 7. IMC exacerbate papilloma formation : (A) Papilloma formation on skin of
TgAC and S100A9tgxTgAC double transgenic mice after TPA treatment for 6 weeks.
Mean and SD of papilloma formation per mouse is shown. Seven mice per group
P=0.0071. (B) Papilloma development in lethally irradiated Tg.AC mice that received
BM from S100A9Tg or WT (control) mice. TPA treatment started three weeks after the
BM transfer. Each group included 4 mice (p=0.035). (C) Papilloma development on
TgAC treated with anti-Gr1 antibody as denoted in the graph. Each group has 5 mice.
Shown is the mean and SD of the number of papillomas per mouse (p<0.001).
73
B
A
C
D
Figure 8. Deficiency of IMC resulted in less papilloma formation:
(A) Expression of S100A9 protein in WT C57BL/6 and S100A9KO mice after TPA or
acetone (vehicle) treatment. Each lane represent 1 mouse. Positive control (PC) are
splenocytes from tumor-bearing mice. (B) The number of Gr-1+ cells in skin of C57BL/6
mice evaluated by IHC. Mice were treated with TPA or acetone for 4 weeks. Each group
included 4 mice. **p<0.001. In all panels mean and SD are shown. (C) Papilloma
formation in WT and S100A9KO mice after DMBA and TPA treatment as indicated in
the graph. Each group composed 7 mice, p=0.003. (D) Number of papillomas in lethally
irradiated C57BL/6 mice that received BM from S100A9KO or WT (control) mice.
Carcinogen treatment initiated 3 weeks after the BM transfer. Each group included 4
mice (p<0.001). Statistical analysis in (C) and (D) is 2 Way Anova.
74
sufficient number of IMC directly from skin for functional tests was not technically
feasible, Gr-1+CD11b+ cells were isolated from BM of WT and S100A9Tg mice, which
had been treated for 4 weeks with TPA. No immune suppressive activity of these cells
in allogeneic mixed lymphocyte reactions (MLR) was detected (Figure 9A). We
evaluated the possibility that more potent pro-inflammatory signals are needed to
convert IMC to MDSC in Tg.AC mice, which could mimic the situation in the skin. We
used i.p. administration of complete Freund’s adjuvant (CFA), a potent pro-inflammatory
stimulus. Although CFA induced a substantial expansion of Gr-1+CD11b+ cells (Figure
9B), these IMC isolated from either WT or from S100A9Tg mice were unable to
suppress antigen-specific CD8+ T cell responses (Figure 9C). Thus, even when
activated with potent pro-inflammatory stimulus, IMC in S100A9Tg did not display
immune suppressive activity.
Since S100A9Tg mice had reduced presence of CD11c + DCs in the skin as
compared with WT mice (Figure 5D), we analyzed in more detail the different
populations of DCs by using flow cytometry. Only slight differences between WT and
S100A9Tg mice were seen in the proportion of subsets of skin DCs (Figure 10A), with
the exception of one population of CD207+CD11b-CD103+ DC, which was significantly
elevated in S100A9Tg mice compared to WT mice (Figure 10A). S100A9Tg had
modestly (p<0.01) reduced number of Langerhans cells (LC) in epidermis (Figure 10B).
To determine if this would translate into decreased migration of DC to draining LN, WT
and S100A9Tg mice were treated topically with acetone vehicle or TPA for 4 weeks
after which the skin was painted with fluorescent dye DDAO.
75
A
B
WT
S100A9Tg
Gr1
Ly6C
CD11b
CD11b
Ly6G
FSC-A
76
C
Figure 9. Accumulative IMC are not suppressive: (A) Suppressive assay of
Gr1+CD11b+ IMC cells isolated from BM of mice treated with TPA. Allogeneic MLR
were performed using CD3-depleted and irradiate FVBN splenocyte as stimulators and
BalbC T cells as responders. Stimulators and Responders were mixed at 1:1 ratio and
IMC were added as indicated. Proliferation was measured by Thymidine incorporation.
(B) The phenotype of cells in spleens of WT and S100A9Tg C57BL/6 mice seven days
after i.p. injection of 100 µg CFA. (C) Effect of Gr-1+ cells isolated from BM of CFA
treated C57BL/6 mice on proliferation of antigen-specific T cells. Gr-1+ cells were mixed
at different ratios with splenocytes from OT-1 mice in the presence of the specific
peptide. Top panel - T-cell proliferation measured in triplicates by [3H]-thymidine uptake.
Bottom panel – the number of IFN-γ producing cells measured in triplicates in ELISPOT
assay. Two experiments were performed with similar results.
The next day skin-draining LN were collected and DDAO+ DCs were counted. We found
significantly lower number of DDAO+ CD86+IAbright migratory DCs in LN of S100A9Tg
mice than in WT mice (Figure 10C).
We wondered whether reduction in DC numbers and migration in S100A9Tg skin
could result in impaired priming of CD8+ T cells. S100A9Tg mice were backcrossed for
9 generations with C57BL/6 mice. DDAO labeled OVA-specific OT-1 T cells were
77
transferred to WT or S100A9Tg C57BL/6 mice pretreated for 4 weeks with TPA. OVA
protein was applied to the same part of the skin as TPA and three days later
proliferation of OT-1 T cells was evaluated in LN and spleen. Robust proliferation of OT1 cells was observed in all mice. No differences were found between WT and
S100A9Tg mice (Figure 10D). These data indicate that despite reduced presence of
DCs in the skin, antigen-specific response was unaffected in S100A9Tg mice. Together
with the data indicating lack of immune suppressive activity of IMC this finding suggests
that immune suppression is not the primary reason for increased tumor formation in
S100A9Tg mice.
IMC recruit CD4+ T cells to the skin. We evaluated the presence of
lymphocytes in the skin of vehicle and TPA-treated mice. No differences between WT
and S100A9Tg mice were found in the presence of B lymphocytes and NK-cells (Figure
11A, B) as well as CD8+ T cells (Figure 11C). Treatment with TPA resulted in
accumulation of CD4+ T cells in the skin that was significantly (p<0.01) higher in
S100A9Tg mice than in WT mice (Figure 11D). In S100A9KO mice, TPA caused
modest increase in skin CD4+ T cells compared to the prominent accumulation
observed in WT C57BL/6 mice (Figure 11E). Skin CD8+ T cells in both WT and
S100A9KO mice were comparably low and unaffected by TPA treatment (data not
shown).
78
A
1010
3+3+
B
79
C
D
LN
Sp
l
48
61.6
44.3
62.2
CD8+CD45.1+
Figure 10. Characterization of DC in the skin of TPA treated mice: (A) Phenotype
of DCs in the skin of WT and S100A9tg mice after TPA treatment measured by flow
cytometry. Three experiments combined are shown. Mean and SD are shown. ***p<
0.001. (B) Langerhans cells in epidermis of mice. On the top is a typical example of
staining. Bar graph on the bottom shows cumulative result of the number of LC per 1
mm2 of epidermis. Each group included 4 mice (mean and SD are shown). ** - p<0.01.
(C). Migration of skin DCs to draining lymph nodes. Dorsal shaved skins of WT and
S100A9Tg mice previously treated with acetone or TPA were painted with DDAO and
24 hours later DDAO+ CD11c+ cells were evaluated in draining lymph nodes by flow
cytometry. Each experiment was performed 3 times. (D) T cells from OT1 mice were
labeled with DDAO fluorescent dye and injected i.v. into TPA treated WT and
S100A9Tg C57BL/6 mice. OVA was applied to the skin 24 hr later and LN and
CD8+CD45.1+ T cells spleens were evaluated by flow cytometry 3 days after the
application. A typical example of the CD8+CD45.1+ T cell proliferation is shown on the
left, and cumulative results (mean + SD) of three mice in each group is shown on the
right.
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A
B cells
B
CD4 cells
D
NK cells
C
CD8 cells
CD4 cells
E
Figure 11. IMC recruit CD4+ T cells to the skin: The proportion of B cells (A) and NK
cells (B) cells among CD45+ hematopoietic cells in WT and S100A9Tg mice treated
with TPA and evaluated by flow cytometry. Each group included three mice. Mean and
SD are shown. C-E. The number of CD8+ (C) and CD4+ (D) T cells in the skin of WT
and S100A9Tg FVB/N mice and CD4+ T cells (E) in the skin of WT and S100A9KO
C57BL/6 mice. The number of cells was evaluated by IHC and counted per mm 2 of
tissue (*- p<0.05, ** - p<0.01). Mean and SD are shown. Each experiment included 5
mice.
The characteristics of CD4+ T cells in skin of TPA treated mice were studied
using flow cytometry. Consistent with the data obtained by IHC, the proportion of CD4 +
T cells among hematopoietic CD45+ cells in skin was significantly (p<0.001) higher in
S100A9Tg mice than in WT mice (Figure 12A). The prevalence of different subsets
within the population of CD4+ T cells in skin was measured using intracellular cytokine
staining. No substantial differences were found between WT and S100A9Tg mice in the
proportion of most CD4+ T-cell populations.
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However, S100A9Tg skin showed higher proportion of IL-22+ and IL-10+ cells,
and FoxP3+CD4+ Treg and a significantly lower proportion of IL-13+CD4+ cells than WT
skin (Figure 12B). No differences were seen between WT and S100A9Tg skin treated
with acetone vehicle alone (data not shown). To investigate the ability of IMC to directly
polarize CD4+ T cells in vitro, naïve CD4+CD62L+ T cells were cocultured with IMC
isolated from BM of TPA treated WT or S100A9Tg mice. No evidence of CD4 + T cell
polarization was found (Figure 12C). Thus, accumulation of IMC in skin of S100A9Tg
mice was associated with substantial increase in the presence CD4 + T cells. Although
IMC did not cause specific polarization of CD4+ T cells, they recruited substantial
proportion of pro-inflammatory T-cell populations. Since accumulation of IMC in the
skin correlated with increased infiltration of CD4+ T cells, we investigated whether
enhanced tumor formation in S100A9Tg mice was mediated by CD4 + T cells.
S100A9Tg; Tg.AC bitransgenic mice were treated with control IgG or CD4-specific
antibody before and during the first three weeks of TPA application. CD4 antibody
significantly (p<0.01) reduced the formation of papillomas in these mice (Figure 12D)
indicating that tumor promoting effect of IMC is mediated via recruitment of CD4 + T
cells.
The mechanism of IMC inducible recruitment of CD4+ T cells. We evaluated
the ability of IMC to attract CD4+ cells in vitro. Supernatants from normal BM derived
IMC had no effect on CD4+ T cell migration. However, IMC activated with LPS or TPA
secreted potent chemotactic activity for CD4+ T cells (Figure 13A). Importantly,
chemotaxis of CD4+ T cells was observed only if T cells were stimulated prior to the
assay (Figure 13A). When IMC supernatants were placed in both chambers during the
82
assay, CD4+ T cell migration was significantly reduced indicating that the effect was
indeed the result of chemotaxis (Figure 13B).
A
CD4+ cells
B
% of CD45+
C
IFN-γ+
IL-4+
IL-17+
D
CD25+FoxP3
+
Figure 12. Characterization of T cell from skin of S100A9tg and WT mice: (A) The
proportion of CD4+ cells among CD45+ hematopoietic cells in the skin of WT and
S100A9Tg mice treated with TPA and evaluated by flow cytometry. Six mice per group
(*** - p<0.001). (B) Intracellular staining of different cytokines in cells isolated from the
skin of WT and S100A9Tg mice treated with TPA. CD4 + cells were gated. Each group
included 3-6 mice(*p<0.05). (C) Polarization of CD4+ T cells in vitro.
Naïve
CD4+CD62L T cell were culture with IMC isolated from BM cells at 1:1 ratio in the
presence of CD3+CD28+ beads for 4 days. Then cells were treated with
PMA/ionomycin in the presence of Golgi-stop for another 4 hours. Intracellular staining
was evaluated within CD4+ population. Total of 5 Mean and SD of 5 experiment are
shown. (D) Papilloma development in CD4 depleted S100A9Tg; Tg.AC bitransgenic
mice. CD4 antibody or IgG (control) treatment was performed as indicated on the graph.
Mean and SD of papillomas per mouse are shown. Each group included 5 mice
(p=0.021).
83
A
B
Figure 13. Activated IMC induce CD4+ T cells migration: (A) Chemotaxis of CD4+ T
cells isolated from control mice to supernatant from IMC. IMC were either stimulated or
not with LPS. T cells were stimulated or not with CD3/CD28 antibody. CD4 + T cells were
placed in the top chamber and supernatants (30% v/v) in the bottom chamber. For
control, LPS was added to the medium in the bottom chamber. Cumulative results of 6
independent experiments are shown (*** - p<0.001) (B) Splenocytes were activated
with anti-CD3 and placed in the top chamber of Transwell plates. Then supernatant of
activated IMC were placed in the bottom or both top and bottom of the Transwell. The
proportion of CD4+ cells were evaluated by flowcytometry. Mean and SD are shown
from 3 experiments. P<0.05
Using RNA isolated from skin, we examined expression of chemokines known to
be involved in the migration of CD4+ T cells. Expression of ccl3 and ccl4 RNAs were
significantly (p<0.05) upregulated in the skin of TPA treated S100A9Tg mice compared
to WT mice, whereas the expression of ccl22 was modestly increased (Figure 14A).
However, no differences in the amount of CCL3 and CCL22 proteins were found in skin
lysates of TPA treated S100A9Tg and WT mice (Figure 14B). In contrast, the amount of
CCL4 in the skin of TPA-treated S100A9Tg mice was significantly higher than in WT
mice. No differences in the amount of CCL4 were observed in spleens, BM, lung, or
liver (Figure 14C). Since skin was the primary site of IMC accumulation in TPA treated
S100A9Tg mice, this data is consistent with IMC as the primary source of CCL4. To
84
verify that IMC are indeed able to produce CCL4, this chemokine was measured in
supernatants from BM IMC isolated from TPA treated WT and S100A9Tg mice. Large
amount of CCL4 was found in supernatants from stimulated IMC (Figure 14D). To verify
the role of CCL4 in CD4+ T-cell recruitment by Gr-1+ myeloid cells, we added
neutralizing CCL4 antibody to IMC supernatants in chemotaxis assay. CCL4-specific
antibody abrogated CD4+ T-cell migration caused by IMC in a dose-dependent manner
(Figure 14E).
To test the role of CCL4 in vivo, WT and S100A9Tg mice were treated for 4
weeks with control or CCL4 antibody concurrent with TPA. Treatment with CCL4
antibody did not affect the presence of Gr-1+ myeloid cells (Figure 15A) or CD8+ T cells
(Figure 15B) in the skin. In contrast, neutralization of CCL4 significantly (p<0.01)
reduced the presence of CD4+ T cells in WT and S100A9Tg skin (Figure 15C). Weekly
treatment of Tg.AC mice with CCL4 antibody during the first 3 weeks of TPA promotion
dramatically reduced the formation of tumors (p<0.01) (Figure 15D).
Similar
experiments were performed in S100A9Tg; Tg.AC bitransgenic mice, that developed
much higher number of papillomas than Tg.AC mice, but the CCL4 antibody still
drastically (p<0.01) reduced the number of tumors (Figure 15E).
Discussion
In this study, we report that IMC directly contribute to skin tumor development via
recruitment of CD4+ T cells. Although the link between inflammation and cancer is well
established,5,32 the contribution of specific cell populations to the initial phase of tumor
85
A
C
B
CCL22
CCL3
CCL4 secreted by IMCs
D
CCL4
E
Figure 14. CCL4 is responsible for the recruitment of CD4+ T cells to the skin: (A)
qRT-PCR analysis of the expression of ccl3, ccl4, ccl5, and ccl22 in skin of WT or
S100A9Tg mice treated with TPA or acetone. Differences in expression of ccl3 and ccl4
were significant *P<0.01. (B) Amount of CCL22 (left) and CCL3 (right) protein measured
by ELISA in lysates of various organs from TPA treated WT and S100A9Tg mice. Mean
and SD from 4 experiments with 3 mice per group are shown. Each measurement was
performed in duplicate and normalized for total protein. Cumulative results from three
experiments are shown. (C) Amount of CCL4 protein measured by ELISA in lysates of
various organs from TPA treated WT and S100A9Tg mice. Mean and SD from 4
experiments with 3 mice per group are shown. Each measurement was done in
duplicate normalized for total protein (** - p<0.01). (E) Neutralizing CCL4 antibody
inhibited migration of activated CD4+ T cells towards supernatant obtained from LPS
stimulated IMC. Concentrations of CCL4 antibody or IgG are shown in the graph. Two
experiments were performed in duplicate (** - p<0.01).
86
development remains unclear. The role of MDSC in tumor immune escape is now well
established. In recent years, evidence pointed to expansion of MDSC in chronic
infection and inflammation. This raised the question as to whether these cells contribute
to tumorigenesis directly or via differentiation to MΦ. MΦs participate in early stages of
cancer and inflammation.408 Because inflammation is so complex, the specific role of
individual cell populations in tumor promotion is difficult to ascertain. S100A9Tg mice
provide a novel experimental model in which tissue accumulation of cells with IMC
phenotype is not caused by infection, trauma, or cancer.
This accumulation was
especially evident in TPA treated mice. The vast majority of myeloid cells accumulated
in skin of S100A9Tg were granulocytic IMC. It is known that activated keratinocytes can
attract granulocytes via release of CXCL8 chemokine.409 This may provide a signal for
initial influx of IMC to TPA treated skin. Though these IMC could be classified as PMNMDSC, they lack immune suppressive activity even after additional stimulation with
various pro-inflammatory stimuli in vitro or in vivo. Consistent with our previous study,167
accumulation of IMC did not result in elevation of MΦ or DC. Although the number of
DCs migrating from skin to LN was decreased, the priming of CD8 + T cells to topical
antigen was not affected. Apparently, the modest decrease in DCs in skin was not
sufficient to inhibit CD8+ T-cell priming. Alternatively, the significantly higher proportion
of CD11b-CD207+CD103+ DCs in S100A9Tg mice may have compensated because
these cells play a major role in cross-presentation of antigens in the skin.410 Collectively,
these data indicate that enhanced papilloma formation in S100A9Tg mice is not due to
immune suppression as typically observed with MDSC in cancer. Our results with BM
transfers and Gr-1-specific antibody depletions in S100A9Tg mice, as well as
87
decreased skin carcinogenesis in S100A9KO mice, demonstrate that IMC were the
primary facilitator of tumor development.
It is known that granulocytic cells may potentially contribute to neoplastic
transformation via multiple mechanisms including increased rate of mutation and
proliferation of cells via release of reactive oxygen species 411-413 activation of
cyclooxygenase-2 in epithelial cells414 and inhibition of nucleotide excision repair in
epithelial cells lines by myeloperoxidase.415-417 In this study we did not evaluate the
malignant conversion of tumor lesions but focused on the earlier events promoting
papilloma formation. We found close correlation between accumulation of IMC and
CD4+ T cells in TPA treated skin. It suggested that IMC could recruit CD4+ T cells.
Indeed, direct in vitro experiments confirmed this hypothesis. Importantly, only activated
IMC were able to recruit CD4+ T cells. IMC recruited different population of CD4+ T
cells. However, S100A9Tg skin had increased (albeit not statistically significant)
presence of IL-22+ pro-inflammatory CD4+ T cells. It was associated with dramatic
decrease in IL-13 producing CD4+ T cells. IL-13 was previously shown to be a negative
regulator of Th17 cells.418
CD4+ T cells and specifically IL-17 and IL-22 producing subsets have been
implicated in tumor development. Deficiency of CD4+ T cells inhibited tumor
development in 2-stage squamous carcinogenesis model,419 and enhanced cancer
progression in methylcholantrene (MCA)-initiated sarcoma development.420 In the
K14HPV16 skin cancer model, with E6/E7 oncogenes expressed in epidermis from
88
A
C
Gr-1
CD4
B
Gr-1
D
E
Figure 15. IMC effect on papilloma formation is mediated via CCL4: (A) S100A9Tg
mice were treated for 4 weeks with TPA and either control IgG or neutralizing CCL4
antibody. Typical example of staining and the number of Gr-1+ cells in the skin (right
panel). (B)The number of CD8+ cells in skin.. (C) Typical example of staining and the
number of CD4+ cells in the skin. Each group included three mice (**p<0.01). Scale bar
= 100µm. (D) The number of papillomas formed in Tg.AC and (E) S100A9Tg;Tg.AC
mice treated with TPA and control IgG or CCL4 antibody as indicated. Mean and SD
are shown. Each group included 4 mice. P=0.001 (D) and P<0.001 (E).
89
keratin 14 promoter, CD4 deficiency drastically attenuated neoplastic progression. 229
Pro-tumorigenic effect of CD4+ T cells were primarily associated with accumulation of
immune suppressive Tregs and pro-inflammatory Th17 cells via release of IL-17, IL-21,
and IL-22.421,422
It was suggested that IL-17 and IL-22 contributes to tumor
development by inducing angiogenesis or directly affecting keratinocyte proliferation
and survival.423,424 IL-17 was also shown to promote accumulation of cells with MDSC
phenotype286 suggesting a feedback mechanism contributing to tumor development. IL17 KO mice in a 2-stage carcinogenesis showed delayed papilloma formation compared
to WT mice.291
The critical role of CD4+ T cells in papilloma formation has been validated in our
study. Our results further reveal a mechanism in which IMC accumulated in skin recruits
large numbers of CD4+ T cells, including pro-inflammatory populations. We identified
CCL4 chemokine as the main factor responsible for IMC mediated recruitment of CD4+
T cells.
CCL4 or MIP-1β is a 69 amino acid member of the β (CC) family of chemokines.
It is produced by various cells, including myeloid cells, and binds to CCR1, CCR5 and
US28 receptors. It was reported that melanoma associated MDSC produce high levels
of CCL4 among other chemokines and in CCR5-deficient mice, growth of transplantable
tumors was delayed.425 Activated CD4+ T cells upregulate CCR5 receptor, allowing
these cells to be recruited to the site of CCL4 production426, which is consistent with our
finding that IMC attracted only activated CD4+ T cells. Although it was reported that
different CD4+ T cell subsets may express unique repertoires of chemokine receptors,
current evidence suggests that CCR5 can be expressed on recently activated cells of
90
any subset,427 consistent with lack of preferential accumulation of specific populations of
CD4+ T cells in the skin of S100A9Tg mice.
Our data suggest sequence of events which may promote tumor formation in skin
and possibly other tissues. Granulocytic IMC accumulate at the site of cutaneous
irritation or inflammation triggered by TPA treatment. Activated IMC release CCL4 that
recruits CD4+ T cells including pro-inflammatory populations. These T cells release IL17 or IL-22 to stimulate proliferation of keratinocytes and promote tumor formation
(Figure 16). The direct effect of IMC on keratinocytes or epithelial cells in other tissues
merits further investigation, as well as the possibility that targeting IMC may benefit
individuals with high risk of cancer development.
Methods
Mice. All animal experiments were approved by University of South Florida
IACUC. Mice were housed in pathogen-free facilities. Tg.AC mice were described
previously426 and obtained from Taconic (Rensselaer, NY). S100A9Tg mice on FVB/N
background167
and S100A9KO mice on C57BL/6 background194 were described
previously. Balb/c, C57BL/6, and FVB/N mice were obtained from National Cancer
Institute (Frederick, MD). OT-1 mice were obtained from Jackson Labs. In
S100A9Tg;Tg.AC bitransgenic mice the presence of S100A9-IRES-GFP transgene was
verified by the expression of GFP in leukocytes from peripheral blood and the v-h-ras
transgene was detected by genomic PCR for the SV40 sequences. In some
experiments, S100A9Tg mice on C57BL/6 background were used after backcrossing
S100A9Tg FVB/N mice with C57BL/6 mice for 9 generations.
91
Figure 16. Illustration of potential mechanism of IMC to induce tumor
development: Upon tumor promoter induction, inflammation is developed and recruits
IMCs from the BM to the targeted tissue. These bone marrow-derived cells can secrete
more inflammatory molecules, such as S100A9 which sustains the inflammatory
environment. More importantly, these IMCs are able to secrete the inflammatory
chemokine CCL4, which is able to mobilize CD4+ T helpers cells into the same targeted
tissue.
Skin Carcinogenesis. Female, aged matched (7-10 weeks old) littermate mice were
used in experiments with Tg.AC and S100A9Tg;Tg.AC mice. Dorsal skin was shaved
and 3 nmoles of TPA (Sigma) in 200 µl acetone vehicle was applied twice a week for 4
or 6 weeks. In the carcinogenesis model in C57BL/6 or S100A9KO mice 100 nmoles of
single dose of 7,12-Dimethylbenz(a)anthracene (DMBA) was applied followed by 10
92
by 10 nmoles of TPA every 24 hours for 12 weeks, as previously described.428
Papillomas were assessed weekly and counted when they reach 1mm for at least 2
weeks. In bone marrow transfer experiments, lethally irradiated (950 rads) mice were
injected i.v. with 106 BM cells from various donors’ mice. Treatments with TPA or DMBA
and TPA started 3 weeks after the transfer and were performed as described above.
Tissue preparation and histology. Slides with frozen tissues were fixed for 10
minutes with acetone and blocked for 60 min with 10% goat serum, 1% BSA and 2.5%
mouse serum in PBS at room temperature. The following primary antibodies from BD
biosciences were used at 1:100 dilutions: Gr1 (RB6-8C5), CD4 (H129.19), and CD8
(53-6.7). The following antibodies from ebioscience were used at 1:50 dilution: CD11c
(N418) and F4/80 (BM8). Biotinylated anti-rat IgG (Vector Labs) or anti-hamster IgG
(Vector Labs) were used as secondary antibodies. Alkaline phosphatase kit and Vector
Red substrate (Vector Labs) were used for visualization of the results. The tissues were
counterstained with hematoxylin and mounted for cell counting. Images were taken by
the digital slide scanner Scanscope (Aperio) and analyzed by Aperio software. Cell
number was calculated per 1 mm2.
Cytokine expression measured by qPCR and ELISA.
For the analysis of
gene expression, the total RNA was extracted with a Trizol reagent (Invitrogen), and
cDNA was synthesized using the High Capacity cDNA Reverse Transcriptase kit
(Applied Biosystems). To detect mouse chemokines, PCR was performed with 2 μl of
93
cDNA, 12.5 μl of SYBR Master Mixture (Applied Biosystems). Primers used for the
different chemokines were the followings: ccl2: forward 5’-CCC AAT GAG TAG GCT
GGA GA-3’ and reverse 5’-AAA ATG GAT CCA CAC CTT GC-3’, ccl3 forward 5’- CCA
AGT CTT CTC AGC GCC ATA-3’and reverse 5'-GAT GAA TTG GCG TGG AAT CTT
C-3', ccl4 forward 5'-TGC TCG TGG CTG CCT TCT-3' and reverse 5'-CTG CCG GGA
GGT GTA AGA GA-3', ccl5 forward 5’-TGC CCA CGT CAA GGA GTA TT-3’ and
reverse 5’-CAG GAC CGG AGT GGG AGT A-3’, ccl9 forward 5′-GAT GAA GCC CTT
TCA TAC TGC -3′ and reverse 5′-GTG GTT GTG AGT TTT GCT CCA ATC -3′ and
ccl22 forward 5′-GTG GCT CTC GTC CTT CTT GC-3′ and reverse 5′-GGA CAG TTT
ATG GAG TAG CTT-3′. Amplification of endogenous β-actin was used as an internal
control. For evaluation of the proteins, BM cells were flushed and tissues were
homogenized in RIPA buffer for protein extraction.
Chemokines’ proteins were
measured by ELISA using the kits PeproTech (Rocky Hill, NJ): CCL3 (900-K125),
CCL4 (900-K278), or CCL22 (900-K197).
Western Blot.
Samples (30ug protein per lane) were subjected to
electrophoresis in 12 % SDS-polyacrylamide gels, and then blotted onto PVDF
membranes. Membranes were blocked for 1 hour at room temperature with 5% dry skim
milk in TBS (20nM Tris-HCl. pH 7.6, 137mM NaCl plus 0.1% (v/v) Tween 20) and then
probed with S100A9 or β-actin specific antibodies (Santa Cruz) followed by secondary
antibody conjugated with horse radish peroxidase. Results were visualized by
chemoluminescence detection using commercial kit (GE Bioscience).
94
Experiments with depletion of cells and neutralization of chemokine in
vivo. Mice were injected intraperitoneal (i.p.) with 250 µg of CD4 (clone: GK1.5,
BioXCell), Gr-1 (clone: RB6-8C5, BioXCell), or 100 µg of CCL4 (R&D System) specific
monoclonal antibodies. Injections were performed once a week for 4 weeks starting one
week before beginning of TPA treatment. Control groups in CD4 and Gr-1 depletion
experiments received Rat IgG2b Isotype (clone: LTF2, BioXCell), in CCL4 neutralization
experiments received goat IgG (RD System).
CD4+ T cell polarization experiments.
Naïve CD62L+CD4+ T cells were
isolated from spleen of FVB/N mice using magnetic beads (Miltenyi) and coculture with
Gr-1+ cells isolated from BM of WT or S100A9Tg mice treated for 4 weeks with TPA.
Cells were activated for 4 days at 37ºC with CD3/CD28 beads. On day 4 cells were
stimulated with 50 ng/ml of TPA and 700 ng/ml of ionomycin for 4 h in the presence of
protein transport inhibitor GolgiStop (BD PharMingen) to prevent cytokine secretion
during the last 3 hr. The cells were harvested followed by intracellular staining with CD4,
IL-17, IL-4, FoxP3, or IFNγ antibodies (all from BD bioscience) and analysis by flow
cytometry.
Skin cell isolation and analysis. Pieces of shaved skin were incubated in PBS
containing 0.2% trypsin for 30 min at 37ºC, cut into small pieces and incubated with 0.8
mg/ml of collagenase in RPMI for 40 minutes at 37ºC. Single cell suspension was
prepared by pressing tissues through 100 µm cell strainer. For the analysis of cell
phenotype, cells were used directly for flow cytometry. Nonspecific staining was blocked
95
by Fc-block (BD) and staining was performed in 1% FBS. The cells were analyzed by
flow cytometry using a LSR II cytometer (BD Biosciences) and FlowJo software (8.6).
Dead cells were excluded by DAPI-staining, and CD45+ cells were analyzed. For the
analysis of intracellular cytokines, mononuclear cells were isolated by density
centrifugation on Ficoll-Paque, washed and plated at 2x106/ml concentration in roundbottom 96 well-plates. Cells were then stimulated with 30 ng/ml TPA and 750 ng/ml
ionomycin for 4 hr, the last 3 hr in the presence of GolgiStop. Cells were stained by
Live-Dead Violet followed by CD45 and CD4 antibodies. After fixation and permeation,
cells were stained for intracellular cytokines or FoxP3 and analyzed by flow cytometry.
Chemotaxis.
Gr-1+ cells were isolated from BM using biotinylated anti-Gr1
antibody and streptavidin microbeads (Miltenyi Biotec). The purity of Gr-1+CD11b+ cells
was greater than 95%. Cells were cultured overnight in serum-free medium (Cellgenix)
at 106/ml, in the presence or absence of 0.5 µg/mL of LPS. Supernatants were collected
after 24 hr and added at 30% v/v to the bottom chamber of Transwell plates (5 µm
pores) in a total volume of 500 µL of serum-free medium. To activate T cells,
splenocytes were cultured with 0.5 µg/ml of anti-CD3 antibody for 3 days in serum-free
medium. One million of stimulated or nonstimulated splenocytes were resuspended in
100 ul of serum-free medium and added to the top chamber of the Transwell. After 4 hr
incubation at 37ºC, CD4+ T cells that passed through the membrane to the lower
chamber were measured by flow cytometry.
96
Langerhans cell staining. Dorsal skin sheets were prepared as previously.429
The epidermal sheets were fixed in acetone for 1 minute, blocked with 1% BSA and
stained overnight at 4ºC with IA/IE-PE antibody (clone 2G9, BD bioscience) at 1:100
dilution. Next day, the sheets were washed stained with secondary antibody and
mounted on slides. The staining was analyzed in SP5 Leica Confocal Microscope.
DC migration and T cell proliferation. Shaved dorsal skin was painted with 0.4
ml of a 1:1 mixture of acetone:DBP containing 250 µM DDAO (Invitrogen). 24 hr later,
draining LNs were collected, treated with 1 mg/ml collagenase in PBS/BSA for 30 min,
and single cell suspensions were stained and analyzed by flow cytometry. For the
analysis of in vitro cell proliferation, 105 splenocytes from Balb/c mice (responders) were
mixed with 105 T-cell depleted and irradiated (20Gy) splenocytes from FVB/N mice
(stimulators) in U-bottom 96 well-plates. Gr-1+ cells from TPA treated S100A9Tg or WT
mice were added into wells at different ratios. On day 4 cells were pulsed with 3Hthymidine (1 µCi/well; GE Healthcare) for 18 hours. 3H-thymidine uptake was counted
using a liquid scintillation counter and expressed as counts per minute (cpm).For the
assessment of in vivo T-cell proliferation in response to antigens, splenocytes and LN
cells collected from OT-1 CD45.1+ mice were labeled with 5 µM DDAO. Eight million of
DDAO labeled OT-1 cells were injected into the tail vein of C57BL/6 (CD45.2 +) WT and
S100A9Tg mice that were pretreated for 4 weeks with TPA. The day after transfer, 10
mg of OVA protein in 500 µL of Ultrasicc gel was applied onto the shaved dorsal skin of
the mice. Three days later skin-draining LNs and spleen were collected. Cells were
stained and analyzed by flow cytometry.
97
Statistical Analysis. Statistical analysis was performed using unpaired 2-tailed
Student t-test with significance determined at p<0.05. For the analysis of papilloma
formation statistical significance of repeated measurements was assessed using twoway ANOVA test.
98
CHAPTER 3. MYELOID-DERIVED SUPPRESSOR CELLS IN THE
DEVELOPMENT OF LUNG CANCER
Rationale
Lung cancer is the leading cause of death among men and women in the United
States.24 It is predicted that lung cancer will caused more deaths than the three most
common cancers combined (colon, breast, and prostate).24
Tobacco smoke is the
principal cause of small cell and non-small cell lung cancer and contributes to more than
80 percent of lung cancer deaths among men and women.24 Tobacco smoke also has
been related to the development of cancer in mouth, lips, pharynx, larynx, esophagus,
pancreas, uterus, colon, bladder and acute myeloid leukemia, besides lung cancer.47
Cigarettes are a complex mix of over 4000 chemical agents that includes lung
carcinogens.37,38 The metabolic activation of this compounds cause genetic alterations
to the DNA resulting in the development of cancer.37 Tobacco smoke also can cause
pulmonary inflammation, which can contribute to the progressive development of lung
cancers. Pro-inflammatory changes have been identified in the lungs of active smokers
but interestingly it also has been notice these transformations in patients after they stop
smoking.430 This can generate smoking-independent oxidant stress thus explaining the
persistence and progression of the disease after smoking has been discontinued.431
The most appropriate model to recapitulate the complexity of the effect of CS on
mice is a smoking chamber where mice are exposed to tobacco smoke for prolonged
99
periods of time. This approach, however, requires a very long exposure of mice to
smoke and results in the development of lung cancer only in a proportion of mice.432 To
achieve the development of cancer, in this model we used urethane as carcinogen to
induce lung cancer.78,433 In our smoking model, CS caused strong accumulation of IMC
lacking immune suppressive activity. To clarify the role of MDSC in the development of
lung cancer we used S100A9 knockout mice. S100A9 was implicated in abnormal
differentiation of myeloid cells in cancer accumulation of MDSC. 434
Using this
experimental model we tested the hypothesis that MDSCs are directly involved in the
development of lung cancer. We found that bonafide MDSCs are important for tumor
progression and their presence negatively affects survival of the mice but further
suggests that tobacco smoking can contribute to the development of lung cancer by
inducing the accumulation of non-suppressive IMC. The presence of IMCs, although
are not suppressive, contributes in the chronic inflammation that has a tumor promoting
effect.
Results
Exposure of mice to cigarette smoke induces expansion of myeloid cells in
mice. To clarify the role of MDSC in lung cancer directly associated with inflammation,
we used CS model in FVB/N mice. First, we asked whether CS alone affected the
presence of different populations of myeloid cells. CS did not change the total number
of leukocytes (data not shown). However, one-month exposure to CS caused a
significant (p=0.01) increase of the presence of Gr-1+CD11b+ cells in PB. Granulocytic
and monocytic subsets were equally increased and their ratio remained unchanged
100
(Figure 17A). Three-month exposure of mice to CS did not further increase the
presence of Gr-1+CD11b+ cells in PB. It remained two-fold (p=0.003) higher than in
control. However, the dramatic re-distribution of myeloid cells towards granulocytic cells
was evident (Figure 17B). Despite some trend to an increase in mice exposed to CS,
the presence of MΦ and DCs was not significantly different from non-treated mice
(Figure 17B).
A
Gr1+CD11b+
Ly6G/Ly6C
ns
F480+
CD11c+
ns
p=0.01
p=0.003
One
Month
B
Gr1+CD11b+
p=0.003
F480+
Ly6G/Ly6C
CD11c+
ns
ns
p=0.03
Three
Months
Figure 17. The proportion of myeloid cells in peripheral blood of mice exposed to
CS: FVB/N were exposed to CS for (A) one and (B) three months. The proportions of
indicated population of myeloid cells were evaluated. Each group included 3 mice.
Myeloid populations in bone marrow (BM), spleens, and lungs were evaluated
after 4 months of CS. Exposure to CS caused significant (p<0.05) increase of the
presence of Gr-1+CD11b+ cells in all tested organs. In spleens this increase was
associated with preferential accumulation of granulocytic cells (Figure 18). The
population of MΦ had not changed in any tested organ, whereas the proportion of DCs
was significantly decreased in lung (Figure 18).
101
Gr1+CD11b+
p=0.03
Ly6G/Ly6C
ns
ns
F4/80+
CD11c+
ns
ns
Bone
Marrow
Gr1+CD11b+
Ly6G/Ly6C
F4/80+
ns
p=0.001
CD11c+
ns
p=0.015
Spleen
Gr1+CD11b+
p=0.04
Ly6G/Ly6C
F4/80+
ns
ns
CD11c+
p=0.03
Lung
Figure 18. The proportion of myeloid cells in different organs of mice exposed to
CS: Mice were exposed to CS for 4 months and the proportions of indicated
populations of myeloid cells were evaluated in different organs. Each group included 3-6
mice. The proportions of cells isolated from lungs were calculated among CD45 +
hematopoietic cells
Thus, CS caused substantial increase in the presence of Gr-1+CD11b+ cells,
which could be represented by MDSC. We evaluated the immune suppressive activity
of these cells. Gr-1+CD11b+ cells were isolated from spleens, lung, or BM and tested in
different assays. These cells did not affect CD3/CD28 inducible T cell proliferation
(Figure 19A) or T cell proliferation in allogeneic mixed leukocyte reaction (Figure 19B).
These results indicate that CS by itself caused substantial accumulation of IMC with
102
granulocytic phenotype. However, these cells lacked immune suppressive activity, and
thus were not bona fide MDSC.
A
B
Figure 19. Gr-1+CD11b+ myeloid cells from mice exposed to CS do not suppress T
cell proliferation: (A) Splenocytes from naïve FVB/N mice were stimulated with CD3
and CD28 specific antibodies in the presence of Gr-1+CD11b+ IMC purified from control
mice and mice exposed to CS at indicated IMC: splenocyte ratios. T cell proliferation
was measured in triplicated by [3H] thymidine incorporation. Each group included 3
mice. (B) Purified IMCs from spleen or lung of smoking or control FVB/N mice were
added at indicated ratios to a mixed lymphocyte reaction, which was set up using
splenocytes from BALB/C mice as responders and irradiated splenocytes from FVB/N
mice as stimulators mixed at 1:1 ratio. T cell proliferation was measured in triplicates by
[3H] thymidine incorporation. Mean and SEM from 3 experiments are shown.
MDSC are critically important for tumor progression. To assess possible
roles of MDSC in the development and progression of lung tumors, wild-type and
S100A9KO FVB/N mice were treated with a single injection of urethane and 4-month
exposure to CS. This treatment resulted in the appearance of a small number of tumor
lesions (Figure 20A). No significant differences in the number of lesions were found
between WT and S100A9KO mice at that time (Figure 20B). However, in contrast to Gr1+CD11b+ cells isolated from spleens of mice exposed to CS alone, the cells with the
103
same phenotype isolated from mice treated with urethane and CS and bearing lung
tumors, had potent immune suppressive activity (Figure 20C). After 4 months of
treatment wild-type mice had significant increase in the presence of MDSC, but not MΦ,
in spleens and lungs (Figure 20D). The number of MDSC in S100A9KO mice was
significantly lower, similar to the level seen in untreated mice (Figure 20D). The number
of MΦ in S100A9KO mice was lower in spleens but not in lungs (Figure 20D). MDSC
isolated from spleens of S100A9KO mice had significantly lower T cell suppressive
activity than MDSC from wild-type mice (Figure 20C).
In a separate set of experiments, we evaluated whether lack of S100A9 had an
effect on the survival of mice treated with urethane and CS. The fate of the mice was
drastically different. All wild-type mice succumbed to the diseases within 5 months after
start of the treatment. In contrast, all S100A9KO mice were alive at that point (p=0.005)
(Figure 20E). Thus, CS, by itself, caused expansion of IMC lacking immune suppressive
activity. Bona fide MDSC accumulated only after the appearance of tumor lesions in
lung. These cells, however, played a major role in tumor progression and survival of the
mice.
Discussion
In this study we investigated the possible contribution of MDSC to the
development of lung cancer. Although ample evidence linked inflammation and cancer,
the nature of the cells directly involved in the process of tumor development remained
unclear. This is especially true for lung cancer. CS is the major cause of lung cancer,
and inflammation is intimately associated with smoking. Since MDSC are shown to be
104
A
C
B
D
Tumor lesions
Spleen
Lung
E
Figure 20. The effect of S100A9 deletion on tumor progression in mice exposed to
urethane and CS: WT and S100A9KO FVB/N mice were treated with a single injection
of urethane (1.5g/kg) and 4 months of CS. A. Typical example of lung tissue staining
with H&E. Scale Bars = 1 µm. B. The number of tumor lesions per lung. C. Immune
suppressive activity of Gr-1+CD11b+ cells isolated from spleens of mice assessed in
allogeneic mixed leukocyte reaction. Each group included 3 mice. Mean and SEM are
shown. The differences between groups: ** - p<0.01; *** - p<0.001. The differences
from “No MDSC” control were significant (p<0.05) in all groups except at 1:4 ratio. D.
The number of indicated myeloid populations in spleens and lungs of mice. * - p<0.05
between the groups. E. Survival of mice of WT (n=6) and S100A9KO (n=7) mice treated
with urethane and CS.
105
an inherent part of chronic inflammation, it was tempting to speculate that MDSC may
be directly involved in the early stages of lung tumor development associated with
chronic inflammation. We expected that in a lung cancer associated with CSinflammation accumulation of MDSCs could happen earlier during treatment. Indeed,
even a one-month exposure of mice to CS led to an increased presence of Gr1+CD11b+ cells in PB, and 4 months of CS caused significant increase of these cells in
lung and spleen. These cells were largely represented by granulocytic subset.
However, contrary to our expectations, these cells lacked immune suppressive activity
indicating that they were not bona fide MDSC. Immune suppressive MDSC
accumulated in these mice only after the development of tumor lesions. These data
suggest that in this smoking model, MDSC accumulation was also the result of tumor
formation. Our results are largely consistent with previous reports demonstrating
association of lung tumor formation caused by urethane injection with accumulation of
Gr-1+CD11b+ cells. However, whether those cells had immune suppressive activity was
not clear.435
These data indicate that CS, despite causing expansion of IMC, was not
sufficient to convert them to MDSC. These results are consistent with two-signal
concept of MDSC accumulation in cancer165 suggesting that the expansion of IMC cells
and their conversion to MDSC phenotype is governed by different factors and signaling
pathways. Some factors involved in expansion of IMC are well characterized (GM-CSF,
M-CSF). However, the mechanism responsible for MDSC conversion is largely unclear
and needs to be determined. Although bona fide MDSC are not present in mice until
appearance of tumor lesions, these cells are critically important for the survival of the
106
mice. We addressed the role of MDSC in the natural history of lung tumor progression
by using S100A9KO mice. S100A9 and its dimerization partner S100A8 proteins play
an important role in myeloid cell differentiation. Their expression is found predominantly
in cells of the myeloid lineage. Differentiation of myelocytes and granulocytes is
associated with increase of S100A8/A9 expression, whereas differentiation of MΦ and
DCs is associated with loss of these proteins.186,436 Lymphocytes and most of the
tissues do not express these proteins. We, and others, have shown that accumulation of
MDSC and inhibition of DC differentiation in cancer were closely correlated with upregulation of S100A8/A9.
S100A8/A9 proteins were implicated in lung cancer
progression. In an early study, S100A8 and S100A9 were shown to be important in
attracting CD11b+ cells to pre-metastatic lung. Neutralizing anti-S100A8 and antiS100A9 antibodies blocked the morphological changes and migration of tumor cells and
CD11b+ myeloid cells.389
Patients with lung cancer overexpressing S100A9 had a
significantly lower 5-year overall survival rate than patients with weak or no expression
of S100A9. Overexpression of S100A9 was an independent factor predictive of poor
disease outcome.437 Consistent with these data, another study also found association
of the expression of S100A8/A9 in stromal cells with negative outcome in patients with
lung cancer.438 Up-regulation of S100A8 and S100A9 genes in bronchial airway
epithelial cell brushings was found to be associated with smoking.439
These results suggested that lack of S100A9 could decrease the accumulation of
MDSC and thus open the opportunity to directly test the role of these cells in tumor
progression. In the urethane/CS model, it did not significantly reduce the number of
initial tumor lesions but S100A9KO mice had much better survival than their wild-type
107
counterparts. Recent study on the model of multiple myeloma has shown that improved
survival of S100A9KO tumor-bearing mice was associated with improved tumor-specific
response of CD8+T cells and was reversed by administration of MDSC.440
Taken together, these data suggest that early stages of tumor development in
CS related lung cancer are associated with accumulation of non-suppressive IMC. This
further necessitates the search for a better way to therapeutically target these cells in
cancer patients. IMC, for their part, may play an important role in tumor development via
mechanisms not involving immune suppression. We recently have found that IMC could
promote the development of skin cancer by recruiting pro-inflammatory CD4+ T cells
(data not shown). These results indicate that IMC and MDSC may be a major factor in
regulation various stages of tumor development and progression and their precise role
needs to be further elucidated.
Methods
Mice.
All animal experiments were approved by University of South Florida
IACUC. Mice were housed in pathogen-free facilities. FVB/N and C57BL/6 mice were
obtained from National Cancer Institute (Frederick, MD). S100A9KO mice on C57BL/6
background were described previously.194
Lung carcinogenesis model. Exposure of mice to CS was performed using a
whole body smoke inhalation system (Teague TE-10C, Teague Enterprises, Davis, CA).
Mice were exposed to an average of 250 mg/m 3 total suspended particulate (TSP) of
CS for 2 hours/day, 5 days/week during 2-4 months using the standard Federal Trade
108
Commission (FTC) method: a 2-second 35 cm3 puff once per minute for a total of nine
puffs. TSPs concentration was characterized daily by collecting particles on 25mm
Pallflex membrane filters. Standard reference cigarettes 3R4F were purchased from the
Tobacco Research Institute (university of Kentucky, Lexington, KY) and used in this
study. At the end of week 3 of smoke exposure, some mice received single
intraperitoneal (i.p.) injection of urethane (1.5 g/kg). CS continued for 3-4 more months.
After 3 or 4 months of exposure to smoke, mice were euthanized and the presence of
myeloid cells and the size and number of tumor lesions were evaluated.
Preparation of single cell suspension and antibody staining. Lungs were
thoroughly minced and digested in RPMI-1640 medium containing collagenase XI (2
mg/ml; Sigma-Aldrich) at 37˚C for 15 minutes. Samples were resuspended in calcium
and magnesium-free PBS-EDTA at room temperature. Red blood cell were lysed with
ACK buffer, washed in PBS-EDTA, and passed through a 50 mm cell strainer. After
single cell preparation cells were counted and stained with following Ab: Gr1-APC,
CD11b-PEy7, F480-PE, CD11c-FITC
Ly6C-FITC and Ly6C-PE. All antibodies were
from BD bioscience.
T cell suppressive activity of MDSC. In experiments with CC10Tg mice on
C57BL/6 background, the effect of MDSC on antigen non-specific (CD3/CD28
antibodies inducible) and antigen-specific (OVA-derived peptide specific OT-1 CD8+ T
cells) proliferation were measured. Briefly, T cells obtained either from wild-type
C57BL/6 mice or from C57BL/6 OT-1 mice were labeled with CFSE and mixed with
109
irradiated (20Gy) syngeneic splenocytes at a 1:4 ratio. MDSC were added at different
ratios and cells were stimulated with either 1 µg/ml CD3 and 0.5 µg/ml CD28 antibodies;
or 0.5 µg/ml OT-1 specific peptide SIINFEKL. Proliferation of T cells was assessed 72
hrs later by flow cytometry. Experiments were performed in triplicates. In experiments
with urethane/CS models on FVB/N background, antigen-specific T cell proliferation
was assessed using allogeneic mixed leukocyte reaction. Briefly, 10 5 splenocytes from
BALB/c mice (responders) were mixed with 105 T cell depleted and irradiated (20Gy)
splenocytes from FVB/N mice (stimulators) in U-bottom 96 well-plates. FACS purified
Gr-1+ cells were added into wells at different ratios. On day 4, cells were pulsed with 3Hthymidine (1 µCi/well; GE Healthcare) for 18 hrs. 3H-thymidine uptake was counted
using a liquid scintillation counter and expressed as counts per minute (cpm).
Evaluation of lung tumors. Whole lungs were flushed with PBS and fixed in
4% paraformaldehyde for at least 24 hrs and paraffin embedded. Paraffin-embedded
lungs were serially sectioned transversally at 4 µm and histologically examined with
hematoxylin and eosin (H&E) stain.
Evaluation of myeloid cells by flow cytometry.
Several populations of
myeloid cells were evaluated in this study: 1) IMC/MDSC defined as Gr-1+CD11b+ cells,
including
subset
of
CD11b+Ly6CloLy6G+
granulocytic
and
CD11b+Ly6ChiLy6G-
monocytic cells; 2) macrophages (MΦ), which were defined as Gr-1-CD11b+F4/80+
cells; and 3) DCs, defined as Gr-1-F4/80-CD11c+ cells.
110
Statistical analysis. Statistical analysis was performed using unpaired 2-tailed
Student t-test with significance determined at p<0.05. For the analysis of survival, LogRank Mantel Cox analysis was used.
111
OVERALL CONCLUSION AND FUTURE THOUGHTS
Participation of inflammation in the development of cancer has been established
for many years. Dr. Rudolph Virchow first described the concept of “lymphoreticular
infiltration” by observing particular cells invading in the emerging neoplastic tissues.7
Later, many studies confirmed that the inflammatory environment, which includes
different cellular and molecular components, plays a crucial role in the processes that
leads to tumor formation. Recapitulation of this idea brought back the interest to study
the process of inflammation that induces cancer.
Many studies in tumor development have implicated myeloid cell populations in
the inflammation processes that lead to cancer development.11,179,441 In the past few
years, many groups deciphered an immature myeloid population capable of
suppressing the normal immune responses against neoplasm.150,442,443
It was
demonstrated in this project, in two different models of inflammation, that the bone
marrow derived immature myeloid cells population can also influence the tumor
development. In the first model, upon inducing inflammation by tumor promoter,
infiltrating IMCs were able to attract CD4 T helper cells by secreting CCL4 chemokines.
This contributes to the concept that not only mature myeloid cells participates in the
inflammatory environment but also immature cells within the myeloid population can
reach the inflammation area at early stages and accelerate neoplastic development. In
the first part of the project the overexpression of S100A9 in mice served as a preamble
112
of the inflammatory environment but additionally could contribute to the abundant
infiltration of IMCs within the targeted site. S100A9 and its partner S100A8 have been
linked not only with different types of cancer but also other health disorders such as
cardiovascular, autoimmune and neurodegenerative diseases among others. 164,186,189 In
a cellular emphasis, this protein previously has been linked directly to accumulation of
Gr1+ cells resulting from the deregulation of DC differentiation in established
tumors.167,428 From this perspective, it was curious to investigate how its overexpression
could influence, at the cellular level, the early stages of carcinogenesis. Most studies
defined MDSCs as one of the principal collaborators in tumor onset and development.
These particular immature cell populations, with Gr1 and CD11b surface markers,
suppress directly the potent immune responses that are built against strange
antigens.146,147 The presence of these cells has been well studied in established tumors
and their environment characterized by inflammation. 162 Recently, an interesting study
revealed that MDSCs have increasing suppressive capacity which depends on the
stage of the tumor. In this study the authors suggest that the suppressive capacity of
these cells augments with the development of the tumor. 444 Thus, the current study
might provide a new perspective of how cellular components, within the early stages of
carcinogenesis, can influence the inflammatory environment that leads to tumor
formation. Naturally, trying to elucidate direct mechanisms of the cellular components in
the tumor environment are the first approach of most studies but is now becoming
equally interesting and important to study the indirect mechanisms of tumor
development which can have a notorious impact in the development of new therapies.
113
The first part of this study showed that IMCs, with markers Gr1+ and CD11b+,
indirectly contribute to tumorgenesis. Importantly, the implication of these cells in the
inflammation process was sustained by the overexpression of S100A9 protein. It was
found that these IMCs migrate from the BM and accumulate at the site of inflammation.
Contrary to the immunosuppressive MDSCs found in established tumors, the Gr1+
CD11b+ cells found in the inflammation area don’t have suppressive capacity and were
characterized with granulocytic phenotype.
Further, it was confirmed that the
participation of adaptive immune cells in the inflammation environment increases the
chances of tumor establishment. In most studies the presence of these cells has been
linked with good prognosis but in recent years, studies confirmed that some CD4+
T helper cells phenotypes can contribute in suppressing immune protection and/ or
inducing more inflammation.226,231,445 Indeed, in this project it is suggested that IMCs
can bring CD4+ T helper cells to the inflammation environment and contribute to tumor
development. It hasn’t been address, in this particular study, which of the known CD4+
T helper cells influence directly the tumor progression. From the recognized CD4+ T
helper cells phenotypes is probably obvious to suggest that in the chronic environment
we will going to find Th17 and/or Tregs regulating the inflammatory responses. In the
case of Th17, known to be a proinflammatory phenotype, can enhance the inflammatory
environment by secreting IL-17 and IL-22.281,446 This idea might be an obvious
suggestion since is also known that IMCs can secrete TGF-β and IL-6, the cytokines
that are required for Th17 differentiation. In many cancerous tumors Th17s are found in
high frequency compared to other T helper phenotypes. 447,448 Sometimes these cells
are the only contributors of IL-17 in the cancer environment which can signify a
114
detriment prognosis of a cancer patient.299 There is a very interesting study suggesting
that human immature myeloid cells isolated from blood of cancer patients can influence
the differentiation of uncommitted CD4 T cells into Th17 or T regulatory cells.305
Although this phenomenon may explain the interaction of IMCs and CD4 T cells in the
tumor microenvironment it requires detailed in vivo experiments to clarify how this might
happens mechanistically. In this study, we provide a step forward that could contribute
to explain how CD4 T cells and IMCs might interact in favor of tumor development.
Alongside with the possibility of trying to elucidate which phenotype of CD4+ cells
could participate during the early stages of carcinogenesis this study also suggest the
participation of CD4 Tregs, the cells that are characterized by their suppressive
activity.309 CD4 Tregs are found often when the tumors are already established and
present one of the mechanisms of tumor escape.449,450 Tregs might participate in
carcinogenesis by secreting pro-inflammatory cytokines. Several studies suggest that
FoxP3-expressing
CD4
Tregs
secretes
IL-17
cytokine
in
the
inflammatory
environment.451,452 This behavior is explained by the plasticity of Th17 and Tregs.
Several
studies
described
this
phenomenon
in
pre-neoplastic
inflammatory
environment.301,305 So it will be interesting to investigate the mechanisms by which the
inflammation process, at early stages of tumor formation, can induce the phenotype
switch of CD4 T cells and what would be the consequences of this phenomenon in the
specific environment. It will be very useful to try to clarify if the CD4 T cells that are
infiltrated in the inflammation environment are contributing directly as IL-17-secreting
CD4 T cells or if these CD4 T cells are the regulatory phenotype that suppresses the
115
early responses of the adaptive immune system which allow the aggressive
development of inflammation and tumor formation.
Another interesting point that was elucidated in this study is that the infiltration of
CD4 T cells was driven by the expression of CCL4 chemokine by IMCs. To our,
knowledge this is the first time that is reported that CCL4 chemokine is secreted by nonsuppressive IMCs and recruits CD4 T cells during the inflammation that leads to tumor
formation. This deciphered event not only can lead to develop a different strategy for
early diagnosis of cancer but also unveiled a possible new pathway of tumor formation.
In cancer related studies chemokines are mostly secreted by stroma cells, tumor cells
and/or mature myeloid cells to attract other inflammatory cells that support cancer
development. They together orchestrate a complex scheme to alter the immune system
to benefit or obstruct the inflammation environment. Thus, chemokines may serve as
the link between both immune systems that together can promote the establishment of
tumors.
Continuing with the same phenomenon, in the second part of this project it was
addressed the effect of the inflammation caused by cigarette smoke in mice. Although
is well known that cigarette smoke can serve as tumor initiator agent we wanted to find
out if MDSCs could participate in this process. It was shown, in the second part of this
study, that there is a significant accumulation of Gr1+ CD11b+ cells in the blood of mice
exposed to cigarette smoke during the first three months of treatment. Interestingly,
when we analyze the function activity of these cells we found that these cells are not
suppressive. Surprisingly, these results were similar to the results found in the
inflammation skin experiments, confirming that these IMCs that accumulate in the
116
previous events of carcinogenesis might contribute to tumor formation by nonsuppressive mechanisms. In the smoking project we had the opportunity to analyze
Gr1+ CD11b+ cells that accumulate in different organs upon carcinogen and smoking
treatment. We found that these cells were suppressive only if tumors were present
suggesting that there could be a mechanism that converts non-suppressive
Gr1+CD11b+ cell into suppressive MDSCs during the early events of tumor formation. It
will be interesting to analyze the mechanisms that are responsible for the ability of these
cells to reach the suppressive state and facilitate the process of tumor formation.
Condamine et al. proposed in a review a “two-signal” stage that these specific IMCs
possibly undergo to accumulate and acquire suppressive characteristics. In this review
he described that the “first signal” is influenced mainly by colony stimulating factors
(CSFs), such as GM-CSF, G-SCF and M-CSF, which promote the accumulation of
these IMCs. To acquire the functional characteristic they could require a “second
signal”. During this following stage pro-inflammatory factors, such as IL-6, IL-1β, IFN-γ,
could promote the upregulation of suppressive enzymes and cytokines which contribute
to the altered immune system.165 This “two-signal” concept could explain the cellular
involvement in this model of chronic inflammation since CSFs and proinflammatory
molecules are overly secreted within this environment and attract IMCs expressing the
corresponding receptors. This suggestion could explain partially how IMCs are recruited
into the inflammation site however, it still remain unclear why these cells could not
become suppressive even though that are located in a constant pro-inflammatory
environment to do so. Although at a molecule level the inflammation environment is
well studied perhaps there is a specific scenario where the cells become insensitive and
117
when the tumor appears the environment changes and induces the functional
immunosuppressive capacity into these cells. More complex investigations are required
to clarify why these cells remain in a non-suppressive state and why are still important
for tumor formation.
We further found out that the absence of S100A9 protein in mice improved
survival of the carcinogen/smoked group compared to its WT counterparts. Recent
studies showed that the presence of this protein is correlated with the infiltration of
myeloid cells and poor prognosis in cancer patients.163,397,437 Our lab established a
direct link in the overexpression S100A9 protein and the accumulation of MDSCs in the
tumor environment.167 So we were interested to know if MDSCs have a role in the
establishment and progression of the tumor. We showed that in the absence of S100A9
protein there were less accumulation of Gr1+ CD11b+ cells in the lung and spleen
compared to WT mice after carcinogen/smoking treatment. This particular result is
somehow expected because it is known that the cells with these surface markers
accumulate in established cancer. Further, and more importantly, we confirmed that
cells isolated from the spleen and lung of WT mice are more suppressive than
S100A9KO mice.
In this smoking project we were able to distinguish the stages of tumor formation
by analyzing the function of IMCs within the lung and spleen. We confirmed that during
inflammation Gr1+ CD11b+ cells accumulate in the lung of smoked mice that could
favor the eventual development of cancer. These cells, in this particular environment,
are not suppressive but they must contribute somehow in the establishment of a chronic
inflammation that leads to cancer. These results open the possibility to improve
118
therapeutic approach by targeting different stages of carcinogenesis. Similarly to the
first part of this project, we utilized S100A9 KO mice to address the contribution of IMCs
in the survival of the smoked mice. We showed that the absence of this protein
improved the resistance to the inflammation caused by the smoking exposure. Since
CS treatment alone wasn’t capable to induce tumor appearance we introduce the lung
carcinogen urethane to study the suppressive capacity of Gr1+ CD11b+ cells found
within the tumor. As expected the IMCs isolated from the lungs with tumor lesions were
suppressive which fits the MDCS functional characteristic.
To recapitulate the whole project we could say that we identified the
characteristics of IMCs at different stages of inflammation-related cancer development
by employing two different inflammation models. These two models have in common
the inflammation-induce tumor development concept but are designed differently, which
allow confirming the results. With the first model, the skin project, we elucidate from the
inflammation stages which immune cells and what possible mechanism they utilized to
promote carcinogenesis. With the smoking project we utilized a different concept of
inflammation and consistently with the skin project showed that the IMCs present in the
inflammatory environment aren’t suppressive. In the skin study we found that these
IMCs draw CD4 T cells with chemokines to the inflammatory environment to promote
tumor development but we didn’t study the actual tumor stages. Moreover, in the
smoking project we took a step forward in the stages of carcinogenesis and confirmed
with previous studies that the IMCs present in the tumor microenvironment itself are
MDSCs. In the smoking study the type of T cells involved in tumor development was not
addressed. However, since in both studies the non-suppressive IMCs are present it
119
would be natural to speculate that CD4 T cells could participate. It is necessary, and
very interesting, to perform further experiments to decipher which of the CD4 T cell
phenotype could participate in the inflammation process that leads to tumor
development in both models. This information not only will contribute to the biology of
cancer development but also could help develop new therapeutic approaches that can
intervene in the early stages of tumor formation characterized by inflammation.
120
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ABOUT THE AUTHOR
Myrna L. Ortiz-Ruiz was born in Mayagüez, Puerto Rico. She did her
undergraduate studies in the University of Puerto Rico, Mayaqüez Campus (UPRM).
While doing her bachelor studies her biology professor, Donato Seguí encouraged her
to venture into science research. Myrna started to work with Donato Seguí in the
Science on Wheels Program and also volunteer in Dr. Rafael Montalvo microbial
ecology laboratory. In 2001, she participated in a basic science summer internship
program in Dr. José García neurobiology laboratory at University of Puerto Rico, Rio
Piedras Campus. All these experiences, in different aspects of science, developed a
strong interest in basic science research.
After finishing her bachelor degree in Industrial Microbiology from UPRM, she
started to work as a microbiology technician in Abbott Biotechnology Ltd located in
Barceloneta, Puerto Rico. Although she was grateful for the opportunity, she felt that her
interest in doing science research was still very strong. Myrna decided to apply for a
master program in molecular medicine from the College of Medicine, University of South
Florida. She earned a Master degree in Molecular Medicine with honors and in 2007
she was accepted in the PhD program in Molecular Medicine from the same institution.
Myrna joined Dr. Dmitry Gabrilovich laboratory at Moffitt Cancer Center in January 2008
Here, she earned her first two first-authored papers and collaborate in others. The ideal
career for Myrna to pursue is one where she can integrate teaching and research
studies in the cancer field without missing important family time.