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The Contribution of Inflammatory Cells to the
Progression of Prostate Cancer
Kia J. Jones
Clark Atlanta University, [email protected]
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ABSTRACT
BIOLOGICAL SCIENCES
JONES, KIA JAVONE
B.S., ELIZABETH CITY STATE
UNIVERSITY, 2007
THE CONTRIBUTION OF INFLAMMATORY CELLS TO THE PROGRESSION OF
PROSTATE CANCER
Committee Chair: Cimona V. Hinton, Ph.D.
Dissertation dated May 2016
In recent years, the causal relationship between inflammation and cancer has
gained wider acknowledgement and acceptance. While various types of immune cells are
involved in the process of inflammation, macrophages represent the major inflammatory
component of many tumors. Derived from circulating monocytes, these cells migrate to
tumor sites in response to molecular cues present within the tumor microenvironment.
Once there, interactions with neoplastic cells shape the differentiation and functional
orientation of macrophages into two phenotypically distinct subsets: the “classically”
activated M1 macrophages and the “alternatively” activated M2 macrophages. The
preeminent paradigm in macrophage-related cancer research is that within the tumor
stoma, macrophages acquire an M2 phenotype characterized by production of proangiogenic factors, ECM degrading enzymes and up-regulation of anti-inflammatory
responses, thereby promoting tumor progression. M1 macrophages, on the other hand are
i
thought to exert anti-tumorigenic effects due to their production of pro-inflammatory
cytokines, and reactive oxygen species (ROS).
While the generation of ROS during immune responses is an important aspect of
immune regulation and host defense, excessive ROS production has been implicated in
the pathogenesis of various degenerative diseases, including cancer. Yet, despite the wellestablished role of M1 macrophages in generating high levels of ROS via NADPH
oxidase (NOX), M1 macrophages are still largely viewed as anti-tumorigenic. Hence, this
study reevaluates the complex interaction between prostate cancer (PCa) cells and tumorassociated macrophages (TAMs), and operates on the premise that PCa cells promote a
pro-tumor microenvironment, denoted by increased inflammation and oxidative stress, in
part, through M1 macrophage-mediated, NOX-derived ROS production. Accordingly,
immunofluorescent analysis of prostate tissue microarrays demonstrated an influx of M1
macrophages in prostate carcinoma. Immature monocytes co-cultured with the poorly
tumorigenic prostate cell line, LNCaP, demonstrated changes in morphology and protein
expression consistent with M1 macrophage polarization. PCa cells co-cultured with M1
macrophages displayed significantly higher intracellular ROS levels. Furthermore, M1mediated ROS generation through NOXs increased prostate cell invasiveness and
anchorage-independent growth. Taken together, results from this study suggest a
potentially novel pro-tumorigenic function of M1 macrophages in early PCa progression,
and aid in understanding the complex role of inflammation in cancer.
ii
THE CONTRIBUTION OF INFLAMMATORY CELLS TO THE PROGRESSION OF
PROSTATE CANCER
A DISSERTATION
SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF DOCTOR OF PHILOSOPHY
BY
KIA JAVONE JONES
DEPARTMENT OF BIOLOGICAL SCIENCES
ATLANTA, GEORGIA
MAY 2016
© 2016
KIA JAVONE JONES
All Rights Reserved
ACKNOWLEDGEMENTS
This dissertation was made possible due to the masterly guidance of my
advisor/mentor, Dr. Cimona V. Hinton. Her patience, motivation, and immense
knowledge were essential in me completing my dissertation. I could not have imagined
having a better advisor for my Ph.D. study. I also owe my deepest gratitude to my
dissertation advisory committee members: Drs. Jaideep Chaudhary, Valerie OderoMarah, Shafiq A. Khan and Michelle Dawson for their insightful comments and
encouragement, but most importantly for the hard questions they posed which not only
challenged me to think critically but also enhanced my research project tremendously. It
is also without question that many thanks are due to my colleagues with whom I entered
into this program with. Those late night study sessions and mock presentations helped
mold me into the researcher that I am today. Also, it has been an honor to work alongside
both past and present members of the “Hinton lab”. Thank you all for your assistance,
inspiration and stimulating discussions. I would also like to thank Clark Atlanta
University, the chair and the staff of the Department of Biological Sciences and Center
for Cancer Research and Therapeutic Development for the financial, academic and
technical support provided. Finally, and most importantly, I would like to thank my
beloved family, of which foremost recognition is given to my wonderful and supportive
parents. This research was supported by the National Institute of General Medical
Sciences (1R01GM106020 and 3R01GM106020); and RCMI (5G12MD007590).
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................... iii
LIST OF FIGURES ........................................................................................................... vi
LIST OF TABLES ............................................................................................................ vii
LIST OF ABBREVIATIONS ........................................................................................... vii
CHAPTER
I.
INTRODUCTION ............................................................................................ 1
II.
LITERATURE REVIEW ................................................................................. 5
Hallmarks of Cancer ......................................................................................... 5
Inflammation: Acute vs. Chronic ...................................................................... 6
Prostate Cancer and Inflammation .................................................................... 8
Prevalence of Prostatic Inflammation ............................................................... 9
Causes of Prostatic Inflammation ................................................................... 11
Inflammatory Cells of the Prostate Tumor Stroma ......................................... 13
Tumor-Associated Macrophages .................................................................... 14
Pro-inflammatory Macrophages and Oxidative Stress ................................... 16
NADPH oxidase expression in Macrophages ................................................. 17
ROS and Prostate Cancer ................................................................................ 18
III.
METHODOLOGY ......................................................................................... 21
Chemicals and Reagents ................................................................................. 21
Human Prostate Epithelial Cell Lines ............................................................. 22
Monocyte Culture and Macrophage Differentiation ....................................... 22
iv
CHAPTER
Monocyte/Prostate Cell Culture ...................................................................... 23
Macrophage Conditioned Media Collection ....................................................23
TMA Immunohistochemistry...........................................................................24
TMA Immunofluorescence ..............................................................................25
Transwell Migration Assay ..............................................................................26
Western Blot Analysis .....................................................................................27
ROS Microplate Assay ....................................................................................27
Invasion Assay .................................................................................................28
Anchorage-Independent Colony Formation Assay......................................... 29
Statistical Analysis .......................................................................................... 30
IV.
RESULTS ....................................................................................................... 32
iNOS detection positively correlated with human PCa tissue samples .......... 32
Prostate cancer cells recruited human monocytes .......................................... 34
Classical stimuli-mediated monocyte differentiation and polarization .......... 36
Macrophage co-culture drives SC differentiation ........................................... 36
Exposure to M1s increases ROS in normal and cancerous prostate models .. 38
M1 macrophages promote prostate tumorigenesis in vitro ............................. 39
V.
DISCUSSION ................................................................................................. 42
VI.
CONCLUSION ............................................................................................... 46
REFERENCES ..................................................................................................................47
v
LIST OF FIGURES
Figure
1.
The Hallmarks of Cancer ........................................................................................ 1
2.
Monocyte to Macrophage Differentiation and Polarization ................................... 2
3.
CXCR4 induces ROS through NOX enzymes........................................................ 3
4.
Prostatitis................................................................................................................. 9
5.
The role of inflammatory cells in the progression of prostate cancer................... 15
6.
Macrophage Engulfing Bacterium into Phagosome….….. .................................. 20
7.
Cell Transformation Assay Principle. ................................................................... 31
8.
IHC analysis detected iNOS in human prostate tissue microarrays ..................... 33
9.
IF analysis of iNOS expression in prostate cancer tissue ..................................... 33
10.
PCa cells recruited human monocytes .................................................................. 35
11.
Classical monocyte-to-macrophage differentiation .............................................. 37
12.
Co-culture drives SC differentiation and polarization .......................................... 38
13.
Exposure to inflammatory macrophages increases ROS in PCa cells .................. 39
14.
M1s promote prostate cancer cell anchorage-independent growth....................... 40
15.
M1 macrophages promote prostate cell invasiveness ........................................... 41
vi
LIST OF TABLES
Table
1.
Human Prostate Cancer Cell Lines ....................................................................... 34
vii
LIST OF ABBREVIATIONS
ANOVA ...............................................................................................Analysis of Variance
APO...................................................................................................................... Apocyanin
BPH ....................................................................................... .Benign Prostatic Hyperplasia
BSA ................................................................................................. Bovine Serum Albumin
CM ......................................................................................................... Conditioned Media
COX ............................................................................................................ Cyclooxygenase
CXCR4................................................................................... Cysteine (C)-X-C Receptor 4
DAPI .................................................................................... 4'-6-diamidino-2-phenylindole
DCF .............................................................................................. 2', 7' –dichlorofluorescein
DCFDA ............................................................... 2',7'-dichlorodihydrofluorescein diacetate
DNA ................................................................................................. Deoxyribonucleic Acid
EGF .............................................................................................. Epidermal Growth Factor
FBS ....................................................................................................... Fetal Bovine Serum
H2O2 ....................................................................................................... Hydrogen Peroxide
HMS ..................................................................................... Hexose Monophosphate Shunt
H&E .................................................................................................Hematoxylin and Eosin
IF ..........................................................................................................Immunofluorescence
IFNγ .........................................................................................................Interferon Gamma
IHC ................................................................................................... Immunohistochemistry
viii
IL-4 .................................................................................................................. Interleukin-4
IMDM .................................................................... Iscove’s Modified Dulbecco’s Medium
iNOS ................................................................................................. Inducible Nitric Oxide
KSFM ............................................................................. Keratinocyte Serum-Free Medium
LPS ......................................................................................................... Lipopolysaccharide
MCSF .....................................................................Macrophage Colony Stimulating Factor
MMP ................................................................................................Matrix Metalloprotease
NAC ........................................................................................................ N-Acetyl Cysteine
NGS....................................................................................................... Normal Goat Serum
NO ..................................................................................................................... Nitric Oxide
NOX ........................................................................................................... NADPH oxidase
Nrf2 ................................................................ Nuclear Factor Erythroid 2–Related Factor 2
O2 ........................................................................................................................ Superoxide
O2-° ........................................................................................................... Superoxide Anion
PBS ............................................................................................ Phosphate-Buffered Saline
PCa ............................................................................................................... Prostate Cancer
PIN ................................................................................... Prostate Intraepithelial Neoplasia
PMA ............................................................................................ Phorbol Myristate Acetate
PSA .............................................................................................. Prostate Specific Antigen
PVDF ................................................................................................... Polyvinyl Difluoride
Pyo ....................................................................................................................... Pyocyanin
RFU ............................................................................................ Relative Fluorescence Unit
RNA .......................................................................................................... Ribonucleic Acid
RNOS ............................................................................... Reactive Nitrogen Oxide Species
ix
ROS .............................................................................................. Reactive Oxygen Species
RPMI ................................................................................. Roswell Park Memorial Institute
RT .......................................................................................................... Room Temperature
SDF1α ............................................................................... Stromal-Derived Factor 1 Alpha
siRNA ............................................................................................... Short Interfering RNA
TAM .................................................................................. Tumor-Associated Macrophages
TMA......................................................................................................... Tissue Microarray
x
CHAPTER I
INTRODUCTION
An association between chronic inflammation and the development of cancer has
long-been appreciated. Over 150 years ago, Rudolf Virchow noted that tumors often
occurred at sites of chronic inflammation.4 Today, the link between inflammation and
cancer is well established (Figure 1); however, many of the molecular and cellular
mechanisms mediating this relationship remain unresolved.5
Figure 1: The Hallmarks of Cancer. The original proposed model to define the six properties that a
tumor acquires included unlimited replicative potential, ability to develop blood vessels (angiogenesis),
evasion of programmed cell death (apoptosis), self-sufficiency in growth signals, insensitivity to inhibitors
of growth, and tissue invasion and metastasis. A combination of recent studies, however, indicate that this
model should be revised to include cancer-related inflammation as an additional hallmark.1
1
2
Figure 2: Monocyte to Macrophage Differentiation and Polarization. This schematic depicts the
differential activation of macrophages and their effect on tumor growth.
A key regulator of the link between inflammation and cancer are tumor-associated
macrophages (TAMs).6 Derived primarily from circulating peripheral blood monocytes,
TAMs form the major leukocytic infiltrate within the microenvironment of many tumor
types. 7 The interaction of monocytes with cancer cells, shapes the maturation and
differentiation of macrophages into two distinct subsets: the “classically” activated M1
macrophages and the “alternatively” activated M2 macrophages.6 These phenotypic states
are classified by their distinct expression of cell surface markers, secreted signaling
molecules, transcription and epigenetic pathways.8 Traditionally, macrophages within the
tumor microenvironment are thought to acquire an M2 phenotype, characterized by their
secretion of proangiogenic molecules and growth factors that promote tumor
progression.9 In contrast, M1 macrophages are regarded as anti-tumorigenic largely due
3
to their production of a myriad of pro-inflammatory mediators, such as nitrogen oxide
(NO) and reactive oxygen species (ROS), as part of an antimicrobial arsenal (Figure 2).
While the generation of ROS during immune responses is an important aspect of
host defense, excessive ROS production is also implicated as the cause of various
degenerative diseases, including cancer. Consistent with this notion, our lab observed
increased ROS generation in prostate cancer (PCa) cells stimulated with the
inflammatory cytokine, stromal-derived factor 1 alpha (SDF1α), and demonstrated that
the primary source of ROS was the membrane-bound enzyme complex, NADPH oxidase
(NOX) 2 (Figure 3).10 We also established that NOX2-mediated ROS production was
critical for the malignant state of prostate cancer cells.10 Interestingly, NOXs are also
major sources of ROS in immune cells.11 Moreover, the isoform, NOX2, has been
specifically identified as the predominant isoform responsible for superoxide generation
Figure 3: CXCR4 induced ROS generation through NOX enzymes. A) C42 cells were pretreated with
NAC or AMD3100 for 1 h, followed by SDF-1α treatment for 3 h. B) C42 cells were pretreated with
apocynin or rotenone for 1 h, followed by SDF-1α treatment for 3 h. Fluorescence measure at 498/522 nm.
4
in macrophages.12 However, despite the well-established role of M1 macrophages in
generating high levels of ROS and other tumor promoting factors, they are still largely
viewed as anti-cancerous.
In sharp contrast to this idea, the paradoxical concept of M1 macrophages
supporting cancer is slowly emerging. Very few studies have reported observations
supporting this claim. Sebens et al. observed that exposure of normal colon epithelial
cells to inflammatory M1 macrophages induced activity of the anti-oxidative
transcription factor, Nrf2, resulting in proteasome activation and protection from
apoptosis.13 Similarly, Comito et al. inadvertently demonstrated that M1 macrophages,
although to a lesser extent than their M2-polarized counterparts, increased invasiveness
of PCa cells and transformed normal human prostate fibroblasts to tumor-associated
fibroblasts.14 Fascinatingly, Fang et al. recently reported that persistent co-culturing of
immortalized normal prostate epithelial cells with macrophages, in the absence of
carcinogens, induced prostate tumorigenesis.15 Therefore, we hypothesized that prostate
cancer cells promote a pro-tumor microenvironment, characterized by increased
inflammation and oxidative stress, in part, through inflammatory M1 macrophagemediated ROS production. This hypothesis was explored through the investigation of the
following specific aims:
Aim 1: To investigate whether pro-inflammatory M1 macrophages infiltrate and mediate
oxidative stress in prostate cancer.
AIM 2: To determine whether M1-mediated ROS generation through NOX progresses
prostate cancer in vitro.
CHAPTER II
LITERATURE REVIEW
Hallmarks of Cancer
Cancer is a complex disease in which cells in a specific tissue are no longer fully
responsive to the signals that regulate cellular differentiation, survival, proliferation and
death. As a result, cells continue to grow and divide in an uncontrolled and indefinite
manner, causing local damage.16 Normal cells have a wide number of intrinsic defenses
to protect against becoming cancerous. Moreover, several injuries (mutations) are needed
to disrupt normal cell growth and survival to form cancer.17 In January 2000 a landmark
paper outlining how cells acquire a cancer-like phenotype was published and detailed 6
key changes or features that make a cancer a cancer. These features are now regarded as
the “hallmarks of cancer”, and represent the fundamental basis of malignancy.18 These
principles are thought to govern all malignancies, simplifying and unifying the huge
variety of diseases that come under the umbrella term ’cancer’. The traits (hallmarks) that
the authors highlight in the paper are: (i) cancer cells stimulate their own growth (selfsufficiency in growth signals); (ii) resist inhibitory signals that might otherwise stop their
growth (insensitivity to anti-growth signals); (iii) resist their programmed cell death
(evading apoptosis); (iv) can multiply indefinitely (limitless replicative potential) (v)
5
6
stimulate the growth of blood vessels to supply nutrients to tumors (sustained
angiogenesis); (vi) invade local tissue and spread to distant sites (tissue invasion and
metastasis).17 However, since the publication of this seminal article the hallmarks of
cancer have been revised to include an additional malignant trait: cancer-enabling
inflammation.19
Inflammation: Acute vs. Chronic
Inflammation can be classified as either acute, occurring over short periods of
time (seconds, minutes, hours, and days), or chronic inflammation, which persists for
extended periods of time.20 The inflammatory response is part of the body’s innate
immune system. It is the body’s normal, biological response when something harmful or
irritating affects a part of the body.21 Although acute inflammation is a healthy
physiological response indicative of wound healing, chronic inflammation has been
directly implicated in a wide range of degenerative human health disorders encompassing
almost all present day diseases.22 In the book, Chronic Inflammation: Molecular
Pathophysiology, Nutritional and Therapeutic Interventions, the authors cover several
pathologies associated with inflammation, including aging, allergies, autoimmune
disorders, atherosclerosis, cancer, chronic wounds, metabolic syndrome, and obesity.23
For many years, chronic and acute inflammatory processes were thought to be
driven by different causes, through the activity of different cells and inflammation
mediators, and to result in quite different outcomes. However, a more modern view
suggests that these processes are interlinked. Moreover, in the setting of acute
inflammation, well-regulated tissue healing can go awry and drive a chronic
7
inflammation process intertwined with fibrosis and related processes. In hepatic,
pancreatic, and gastrointestinal issues, among others, this pro inflammatory pro-fibrotic
environment can stimulate carcinogenesis which in turn, can lead to an altered
immune/inflammatory milieu.23
Acute inflammation is the early stage of inflammation and presents with
immediate signs and symptoms such as pain, heat, redness, swelling, and loss of function.
These symptoms are due to the increased blood flow and increased permeability of the
vasculature which is responsible for bringing neutrophils to the affected site as rapidly as
possible. This protects the injured areas from further harm while the body goes into
overdrive to fend off bacteria, viruses, pathogens, damaged cells, or other irritants. It not
only kills off the invaders, but damaged tissue as well, until your body wins the battle.
Without inflammation, wounds and infections would never heal.23
Prolonged inflammation, known as chronic inflammation, leads to a progressive
shift in the type of cells present at the site of inflammation and is largely characterized by
an influx of macrophages. These cells, along with other cellular mediators, contribute to
the simultaneous destruction and healing of the tissue during the inflammatory process. It
can result from failure to eliminate an acute inflammation stimulus, an autoimmune
response, and/or persistence of a chronic irritant of low intensity.23 Unfortunately,
chronic inflammation will not typically produce symptoms until actual loss of function
occurs. This is because chronic inflammation is low-grade and systemic, often silently
damaging your tissues over an extended period of time. This process can go on for years
undetected, until a disease suddenly manifests.23
8
About 20% of all human cancers are caused by chronic infection or chronic
inflammatory states.24 In regards to prostate cancer, it has been proposed that exposure to
environmental factors such as infectious agents and dietary carcinogens, and hormonal
imbalances lead to injury of the prostate and to the development of chronic inflammation
and regenerative ‘risk factor’ lesions, referred to as proliferative inflammatory atrophy
(PIA).24 The development of new experimental animal models coupled with classical
epidemiological studies, genetic epidemiological studies and molecular pathological
approaches, has shed more light on the role of inflammation in prostate cancer, and has
contributed to the advent of new strategies to prevent the disease.
Prostate Cancer and Inflammation
The prostate is a walnut-sized gland located between the bladder and the penis,
anterior to the rectum. The urethra runs through the center of the prostate, from the
bladder to the penis, allowing the outflow of urine from the body. The primary function
of the prostate is to secrete fluids that nourish and protect sperm. During ejaculation, the
prostate squeezes this fluid into the urethra, whereby it is then expelled with sperm as
semen.25 Inflammation is very common within the normal adult prostate,26 and
researchers now appreciate a potential link between chronic prostatic inflammation and
prostate carcinoma (Figure 4). Histological studies have reported unexplained acute and
chronic inflammation, and inflammation-associated lesions, in biopsy specimens of
prostate cancer tissues.24 Moreover, analysis of surgically resected tissues of patients with
benign prostate hyperplasia (BPH) or prostate cancer revealed significant variations in
the expression of several signaling components involved in the inflammatory cascade,
9
including chemical signaling cytokines and their cognate chemokine receptors,
suggesting a connection between inflammatory signaling and prostate carcinoma.27
Prevalence of Prostatic inflammation
There are multiple different lines of evidence suggesting that inflammation is very
common within the adult prostate. Prostatitis is a heterogeneous and complex entity
which the National Institutes of Health (NIH) consensus classification refers to as chronic
Figure 4: Prostatitis. Reprinted from Herballove.com., Retrieved October 28, 2015, from
http://www.herballove.com/guide/your-guide-prostatitis. Copyright © Herballove. All Rights Reserved.
prostatitis /chronic pelvic pain syndrome (CPPS).28 CPPS is divided into the following
four categories, the first three of which relate to men with symptoms of disease: (i) acute
bacterial prostatitis; (ii) chronic bacterial prostatitis; (iii) chronic prostatitis ⁄ CPPS; and
(iv) asymptomatic inflammatory prostatitis.28 Bacterial prostatitis accounts for only an
estimated 5–10% of prostatitis cases, with the most commonly implicated
microorganisms being Escherichia coli (E coli) and Enterococcus spp.29, 30 In terms of
10
symptomatic prostatitis, it is estimated that up to 16% of men in the US population are
afflicted at some time in their life.29, 31
The prevalence of asymptomatic prostatic inflammation (i.e. histological
prostatitis) appears to be in fact much higher, as evidenced by studies examining men
who undergo biopsy for prostate cancer due to elevated prostate-specific antigen (PSA)
levels and test negative for cancer,32,33, 34, 35 autopsy studies36 and findings from
transurethral resections for benign prostatic hyperplasia (BPH).37 A recent example of
this stems from results published from the baseline data of the REDUCE (REduction by
DUtasteride of prostate Cancer Events) trial, where 80% of patient biopsies were found to
have some degree of Inflammation.38 Similarly, results from a prospective randomized
controlled trial of 328 men with PSA levels between 2.5 and 10 ng/ml and normal digital
rectal examination (DRE) indicated that more than 45% of the patients had leucocytes in
expressed prostatic secretions (EPS).34 Finally, histological specimens of prostate cancer
tissue frequently exhibit unexplained acute and chronic inflammation and inflammationassociated lesions.24
Evidence suggests there is also a racial and geographical difference in the
prevalence of prostatic inflammation in adult men, which falls in line with the geographic
distribution difference in prostate cancer incidence. For example, studies have reported
an increased incidence of inflammation in biopsy specimens39 and increased expression
of immune-related genes in tumor tissues 40 from African American men compared to
European American men. Also, recent findings from an autopsy study done by Joshu et
11
al. revealed less inflammation in the prostates of Asian men as opposed to European
American men.26
Causes of Prostatic Inflammation
Multiple bacterial species are known to infect the human prostate and induce
inflammation; many of which have been identified from studying patients with bacterial
prostatitis.26 Interestingly, in the Ugurlu et al. study previously mentioned ,34 the patients
with leucocyte-positive EPS were randomized into antibiotic (levofloxacin), antiinflammatory (naproxen sodium) and control treatment groups. Only the antibiotictreated patients exhibited a significant decrease in PSA levels, suggesting a potential
contribution from an unrecognized prostatic infection.26
In addition to the most commonly implicated microorganisms in bacterial
prostatitis (E. coli and Enterococcus spp), organisms such as Pseudomonas spp., Proteus
mirabilis, Klebsiella spp. and Serratia spp. have also been identified.29, 30 Several sexually
transmitted organisms have also been implicated in bacterial prostatitis and/or prostatic
inflammation including Chlamydia trachomatis, Gonococcal organisms, Trichomonas
vaginalis and Treponema pallidum.41 Mycoplasma spp. have also been implicated in
chronic prostatitis.42 43 Studies attempting to define a potential correlation between
prostatitis and prostate cancer risk have reported both positive and negative results.44 A
study in 2010 performed in a large, multiracial and ethnic cohort as part of the California
Men’s Health Study (CMHS) found an increase in risk for prostate cancer in men with a
history of prostatitis and long duration of prostatitis symptoms.45 This study also found
that a self-reported history of sexually transmitted disease (STD) was not associated with
12
overall prostate cancer risk; however, Latinos reporting a history of STDs had an
increased risk of prostate cancer compared to Latinos with no STD history.45
Furthermore, non-US-born Latinos were found to have a greater risk of prostate cancer
associated with STD history than US-born Latinos.45 Although the authors report that this
study could have potentially been confounded by detection bias (e.g. men with
symptomatic prostatitis may seek medical attention, which may in turn lead to a greater
chance for testing and incidental detection of prostate cancer), the association between
prostatitis and prostate cancer risk certainly remains an important area for further
research.
There are also several lines of evidence that support a potential role for
asymptomatic (i.e. subclinical) prostatic inflammation caused by infectious
microorganisms and prostate cancer development.26 An organism of particular interest in
this respect is E. coli. Apart from being one of the most frequently isolated
microorganisms from patients with bacterial prostatitis, E. coli has also been identified in
both BPH and prostate cancer tissues using both culture-dependent and cultureindependent molecular techniques.46, 47 In mouse models, infection of the prostate with
uropathogenic strains of E. coli (UPEC) has been reported to induce epithelial
proliferation and reactive hyperplasia,48 dysplasia and oxidative DNA damage49 and a
marked reduction of the haploinsufficient prostate cancer tumor suppressor, NKX3.1.50 A
recent study in Wistar rats described prostatic epithelial hypertrophy and atrophy in
response to UPEC E. coli infection, with a transient upregulation of ErbB2 [human
epidermal growth factor receptor 2 (HER2 ⁄ neu)].51 Various strains of UPEC E. coli
13
produce a number of virulence factors, such as cytotoxic necrotizing factor 1 (CNF1),
which has been shown to promote tissue damage in a rat model of prostatitis.52
Intriguingly, in a recent study examining E. coli isolates from patients presenting with
acute bacterial prostatitis, more than 70% of isolates were found to express colibactin and
one strain carried the cyto-lethal distending toxin (CDT) gene cluster.53 The authors of
this study postulate that the production of genotoxic toxins by E. coli implicated in acute
bacterial prostatitis could contribute to subsequent carcinogenesis and potentially explain
the epidemiological data suggesting an increased risk for prostate cancer with previous
history of prostatitis.53
Additional microorganisms (both bacterial and viral) have been implicated in
stimulating prostatic inflammation that may contribute to the development of prostate
carcinogenesis. Moreover, it is believed that recurrent microbial infections of the prostate
resulting in the formation of lesions are due to the persistent secretion of free radicals and
inflammatory cytokines by infiltrating leukocytes. As a result, leukocytic infiltrates have
been identified as key players in prostate cancer development.49
Inflammatory Cells of the Prostate Tumor Stroma
The inflammation identified histologically in prostate cancer tissues is most
commonly chronic, being chiefly comprised of lymphocytes as well as macrophages
(Figure 5), and less frequently of plasma cells and eosinophils.26 Acute inflammation is
present to a lesser extent and is comprised primarily of neutrophils.26 Just as the stimuli
for prostatic inflammation are largely yet to be defined, our understanding of prostate
immunobiology is still relatively poor. Over the past few years, however, there have been
14
several recent advances in the characterization of the inflammatory cell types infiltrating
the prostate.26
The normal prostate contains a heterogeneous inflammatory cell population of
stromal and intraepithelial T and B lymphocytes, mast cells and macrophages,54 which
are also present in chronically inflamed prostate tumors.26 Tumor-associated
macrophages (TAMs) represent the major inflammatory components of the tumor stroma
and direct multiple aspects of neoplastic growth.55 Derived from monocytic precursors in
the blood and bone marrow, TAMs are mononuclear phagocytes, which once localized in
tissues, acquire specialized functions depending on the requirements of the tissue. For
instance, Comito et al. demonstrated that secretion of the chemokine CCL2 from PCa
cells facilitated monocyte recruitment, TAM differentiation and polarization to a specific
phenotype.14 56
Tumor-Associated Macrophages
TAMs are traditionally sub-classified as M1 (classically-activated macrophages),
or M2 alternatively activated macrophages. These phenotypic states are characterized by
their distinct expression of cell surface markers, secreted signaling molecules and
transcription and epigenetic pathways.8 TAMs in murine and human tumors are thought
to typically display an M2-like phenotype,57 which promotes tumor growth, tissue
remodeling, debris scavenging, angiogenesis and suppression of adaptive immunity. M2
macrophages are generally distinguished by their low production of pro-inflammatory
cytokines, and high production of anti-inflammatory cytokines.58 M2 macrophages are
15
diverse, but are all generally involved in T helper 2 (Th2) responses, have immune
regulatory function, orchestrate encapsulation and containment of parasites, and promote
tissue repair, remodeling, and tumor progression;59 all of which favor tumor development
and progression.
Figure 5:
The role of inflammatory cells in the progression of prostate cancer. Recruitment of
inflammatory cells to the prostate can occur under a variety of physiological and pathophysiological
conditions, such as wound, infection, prostatitis, and cancer. Genetically altered prostate epithelial cells,
under the influence of resident fibroblasts, inflammatory cells (such as macrophages and lymphocytes), and
endothelial cells progress further through additional genetic changes triggered by reactive oxygen species
(ROS) or reactive nitrogen species (RNOS). The genetically unstable prostate cell clusters can form PIA and
then proceed to PIN before becoming prostate cancer cells with increased malignant potential.2
16
M1 macrophages are referred to as “killer” macrophages due to their potent
microbicidal and tumoricidal activity.60 These specialized phagocytic cells attack foreign
substances, infectious microbes and cancer cells through destruction and ingestion.
During the initial leukocyte migration phase, M1 macrophages produce a myriad of proinflammatory mediators, such as reactive oxygen species (ROS), as part of an
antimicrobial arsenal. M1s also secrete matrix metalloproteinases (MMPs) that degrade
the extracellular matrix, allowing infiltration of additional inflammatory cells to sites of
tissue injury.60 Should tissue-damaging irritants persist, activated M1s further exacerbate
the inflammatory response by recruiting various adaptive immune cells, thereby
generating substantial tissue damage.60 Once the inflammatory stimulus is eliminated,
M1-mediated inflammatory activation diminishes. On the contrary, a protective role in
tumorigenesis has also been described for M1 macrophages, whereby they antagonize the
suppressive activities of M2 macrophages.60
Pro-inflammatory Macrophages and Oxidative Stress
During acute inflammatory responses, ROS molecules are produced in large
quantities by M1 macrophages and are vital to the destruction of invading pathogens and
foreign materials. These highly reactive oxygen intermediates include superoxide,
hydrogen peroxide, singlet oxygen and nitric oxide.60, 61 Macrophage influx in target
tissues during an immune response generates copious amounts of ROS, and as a result,
leads to tissue damage.62 For instance, in rats administered large doses of vitamin A,
which activated macrophages in the liver to produce ROS, tissue injury was significantly
increased.63
17
The notion that oxidative stress is important for driving prostate cancer
progression is bolstered by studies demonstrating that the consumption of certain types of
dietary antioxidants is associated with reduced prostate cancer risk.24 Excessive,
unregulated generation of ROS leads to oxidative stress. Biomolecules such as lipids,
proteins, and deoxyribonucleic acid (DNA) are all targets for modification by ROS with
diverse pathologic consequences, ranging from cell death (necrosis, apoptosis), to cell
survival, (proliferation, invasion and migration). Accordingly, oxidative stress is
implicated in the pathology of several human diseases, including cardiovascular disease,
rheumatoid arthritis and cancer. We have demonstrated differential pathological
responses to the ROS molecule hydrogen peroxide (H2O2), where treatment of low grade
prostate cancer cells with H2O2 resulted in rapid apoptosis,64 and treatment of high grade
prostate cancer tumors with H2O2 rapidly increased expression of the chemokine Gprotein coupled receptor, CXCR4, resulting in rapid cell migration and invasion through
bone endothelial cells,65 independent of its ligand, SDF1α.65 We’ve also demonstrated a
reciprocal relationship between CXCR4 and ROS, where treatment with SDF1α
increased ROS generation specifically from the membrane associated enzyme complex
NOX2 in prostate cancer cells, which led to enhanced CXCR4-mediated migration.10
Moreover, other groups support our observations and have identified NOX enzymes as
the primary source of ROS in prostate tumors.10, 66
NADPH Oxidase Expression in Macrophages
ROS produced in tissues are derived from two major sources: (i) the
mitochondrial electron transport chain and/or (ii) NADPH oxidase enzymes (NOXs).67
18
While mitochondria generate ROS indirectly as a by-product of cellular metabolism,
NOX enzymes produce ROS directly; to date ROS production is the only known
biological function of NOX enzymes.11 Initially observed in leukocytes, the NOX family
of enzymes is comprised of 7 known members: NOX isoforms 1-5, DUOX1 and
DUOX2.11 All members of the NOX family consist of 6 transmembrane domains, a
NADPH-binding domain and a FAD-binding domain. Despite their structural similarities,
NOX isoforms display distinct biochemical characteristics and subcellular localizations.68
Activation of NOX enzymes require translocation of the cytosolic regulatory proteins to
the plasma membrane-bound flavocytochrome b558, which, once collectively assembled,
generates superoxide by transferring an electron from NADPH to oxygen, resulting in
ROS generation (Figure 6) and diverse biological functions ranging from apoptosis to
mitogenic signaling.11 Macrophages generate ROS via NOX enzymes, and NOX2 is the
predominant form expressed in macrophages of all types.11
ROS and Prostate Cancer
Prostate cancer is commonly associated with a shift in the antioxidant-prooxidant
balance towards increased oxidative stress. Previous studies highlighted the altered
prooxidant-antioxidant status in prostatic tissue of man, rat and also in cell lines, where
the imbalance between these antagonist played a major role in the initiation of prostate
carcinogenesis.69 However, there is very little idea about the cause of this imbalance.
Androgens are considered to be the most powerful candidates that regulate ROS balance
in the prostate, though the mechanistic relation between androgen status and redox
homeostasis in the prostate is not proven.70 Tam et al.71 in this context indicated that
19
replacement of androgens reduced the oxidative stress level by down-regulating NOX
expression, thereby bringing the antioxidant level to normalcy. Besides androgen, the
transcription factor erythroid 2p45 (NF-E2)-related factor 2 (Nrf2) mediates the
expression of key protective enzymes through the antioxidant-response element (ARE) in
prostate cancer.72,73 Recent studies suggested that Nrf2 and several of its target genes are
significantly down-regulated in human prostate cancer and as a result, cells were
continually exposed to increased oxidative stress and may have resulted in their
progression to metastatic disease.74 Another major component involved in the
maintenance of redox balance in the cell is the Glutathione oxidation-reduction system.
Somatic mutations, causing inactivation of the Glutathione S-Transferase gene (GSTP1)
have been identified in almost all the prostate cancer cases examined by Nelson and
colleagues.75 Therefore, the sensitive balance between the oxidant and anti-oxidant
components of the cells and their regulatory mechanisms seem to play a major role in
developing a malignant state in prostate tissue.
20
Figure 6: Macrophage engulfing a bacterium into a nascent phagosome. NADPH oxidase assembles
and begins to function before the phagocytic cup has sealed. In eosinophils, macrophages, and neutrophils
stimulated with soluble agonists, most NADPH oxidase assembles at the plasma membrane. The entire
system is driven by NADPH oxidase activity. Electrons from cytoplasmic NADPH are translocated across
a redox chain to reduce O2 to superoxide anion (O2−•) inside the phagosome or extracellularly. For each
electron removed from the cell, approximately one proton is left behind. Thus NADPH oxidase activity
tends to depolarize the membrane. NADPH is regenerated continuously by the hexose monophosphate
shunt (HMS) during the respiratory burst.3
CHAPTER III
METHODOLOGY
Chemicals and Reagents
The following reagents and human antibodies were from Thermo Fisher
Scientific: Alexa Fluor 488 goat anti-mouse (A11001), Alexa Fluor 488 goat anti-rabbit
(A-11008), Alexa Fluor 594 donkey anti-goat (A-11058), Alexa Fluor 594 goat antirabbit (A-11037) and Keratinocyte-SFM 1X (17005-042). Human recombinant SDFlα
(300-28A), IFNγ and IL-4 were purchased from PeproTech. Human antibodies against
COX-2 (sc-795), PSA (sc-7638) and DAPI (sc-3598) were from Santa Cruz. Apocynin
(sc-203321) was also purchased from Santa Cruz Biotechnology, Inc.The following
reagents and human antibodies were from Sigma Aldrich: Triton X-100 (T8532); antiαTubulin (T5168); thymidine (T1895-5G); hypoxanthine (9636-5G); 2 mercaptoethanol
(M3148-25ml); LPS (L5293) and PMA (P1585-1MG). iNOS (GTX31048) and CD163
[EDHu-1] (GTX42364) were purchased from Genetex, Inc. Hemacolor rapid staining
solution (111661) was purchased from EMD Millipore. COX1 human antibody (ab695)
and cellular ROS/superoxide detection assay kit (ab139476) were from abcam. Sudan
Black B (0593-25G) was purchased from AMRESCO. RPMI 1640 (10-040CV) was from
Corning. CytoSelect 96-Well Cell Transformation Assay, Standard Soft Agar (CBA-130)
kit was purchased from Cell BioLabs, Inc.
21
22
Human Prostate Epithelial Cell Lines
The normal prostate epithelial cell line, RWPE1 (CRL-11609), and prostate
cancer cell lines LNCaP (CRL-1740), DU145 (HTB-81) and PC3 (CRL-1435) were all
obtained from American Type Culture Collection (ATCC) and maintained per
manufacturer’s instructions. In brief, RWPE1 cells were cultured in complete
Keratinocyte Serum-Free Medium (KSFM; supplemented with 0.05 mg/ml bovine
pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF). Prostate cancer cell
lines were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640; supplemented
with 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% antibioticantimycotic and 1% L-glutamine). All cells were maintained in a 37 °C, 5% CO2
incubator. Serum starvation conditions consisted of culturing in reduced serum medium
(unsupplemented KSFM or phenol-free RPMI containing 1% L-glutamine and 0.2%
FBS) for 24 hours.
Monocyte Culture and Macrophage Differentiation
The human peripheral blood mononuclear cell line, SC (CRL-9855), was obtained
from ATCC and maintained at a concentration between 2 x 105 and 1 x 106 cells/mL in
Iscove's Modified Dulbecco's Media (IMDM) supplemented with 10% FBS, 0.05 mM 2mercaptoethanol, 0.1 mM hypoxanthine and 0.016 mM thymidine. To generate resting
macrophages (Mϴ), SC cells were cultured in 60ng/mL phorbol myristate acetate (PMA)
for 6 hours. Subsequent M1 or M2 polarization was achieved via treatment for an
additional 62 hours with lipopolysaccharide (LPS; 0.1μg/ml) and interferon gamma
(IFNγ; 20ng/ml) or interleukin 4 (IL-4; 20ng/ml), respectively.
23
Monocyte/Prostate Cell Co-culture
For monocyte/prostate cell co-culture, seed 1 x 106 (1,000,000) prostate epithelial
cells (RWPE1, LNCaP, DU145 and PC3) each into accompanying 0.4-µm pore transwell
inserts (Corning Cat No. 3412) in 2 mL of RPMI (supplemented with 0.2% FBS) or
KSFM (supplemented with 0.2% BPE; RWPE1) and incubate for 24 hours in 37°C/5%
CO2 incubator. After 24 hours, without removing inserts, seed 1 x 106 SC cells into the
bottom well of each well in 1 mL of serum-free IMDM. Maintain and monitor cocultures for an additional 48 hours.
Macrophage Conditioned Media Collection
SC cells were seeded at a concentration of 4 x 105 (400,000) cells/well in
complete IMDM in a 6-well plate format. Macrophage polarization was achieved by
adding 60ng/ml PMA to each well followed by incubation at 37°C in 5% CO2 incubator
for 6 hrs. After 6hrs, plates were retrieved from incubator and (without removing existing
well contents) M1 or M2 polarizing components were added (LPS; 0.1μg/ml and IFNγ;
20ng/ml or IL-4; 20ng/ml, respectively) and incubated for an additional 66 hours. Next,
cells were pretreated for 1 hour with desired experimental conditions (apocyanin;
300µM), immediately followed by aspiration of all well contents. Serum-free IMDM was
added to each well and returned to incubator for 48 hrs allowing medium conditioning to
take place. After 48 hrs plates were retrieved from incubator and conditioned media
collected and transferred to prelabeled/precooled 15ml conical tubes on ice. To clarify
CM (remove cells and debris) samples were centrifuged at 2,000 x g at 4˚C for 15 min.
After centrifugation, CM was removed being sure not to disturb any sediment, and
24
transferred to newly labeled 15ml conical tubes. Centrifugation step was repeated and
CM was placed on ice for same day use. Alternatively, if CM was not needed for same
day use, it was stored at -20˚C for up to 48 hours.
TMA Immunohistochemistry
Immunohistochemical (IHC) analysis was performed on a prostate disease
spectrum tissue array (PR8011; Biomax) ranging from normal to high grade metastatic
tissues. The array consisted of 80 total tissue cores including adenocarcinoma, metastatic,
hyperplasia, chronic inflammation, adjacent normal tissue and normal tissue. Each
individual core had a diameter of 1.5 mm and a thickness of 0.5 um. Briefly, formalinfixed, paraffin-embedded specimens were retrieved in a down-graded series of xylene
and ethanol incubations. Antigen retrieval was carried out using the Biocare Digital
Decloaking Chamber and Reveal Decloaker RTU per manufacturer’s instructions. Native
peroxidase activity was neutralized via incubation in 0.3% hydrogen peroxide for 15 min
at room temperature (RT), followed by a 1X PBS wash in a humidified chamber.
Specimens were then blocked with blocking solution (5% normal goat
serum/Tris-buffered saline/Tween-20; TBST) for 30 min. Inducible nitric oxide (iNOS)
was detected with a rabbit anti-human iNOS polyclonal antibody (Genetex; 1:1000) in
blocking solution overnight at 4°C, followed by a biotinylated affinity purified goat antirabbit IgG (H+L) secondary antibody (Vector .Laboratories; 1:1000), in blocking solution
for 30 min at RT. Specimens were washed thoroughly between incubations, and avidin +
biotin reagent (ABC solution) was added at a 1:200 dilution for 30 minutes at room
temperature. Next, tissues were developed in diaminobenzidine (DAB) (Vector
25
Laboratories) for 3 min at RT, and nuclei counterstained with Meyer's hematoxylin for 13 min. Following counterstaining, specimens were rinsed in deionized water for 5 min
and subjected to dehydration in graded-up series of ethanol followed by incubation in
xylene, prior to mounting on slides. A negative control tissue sample was prepared by
incubating in biotinylated affinity purified goat anti-rabbit IgG (H+L) antibody, only, as
described above. The specimens were analyzed and photographed using the LSM 700
(Zeiss) confocal microscope.
TMA Immunofluorescence
Prostate tissue microarray’s (TMAs) (PRC481; US Biomax, Inc.) representative
of normal, benign prostate hyperplasia (BPH), chronic prostatitis, low, moderate and high
grade carcinoma lesions were analyzed via immunofluorescence (IF) using iNOS rabbit
polyclonal antibody (1:100 dilution; Genetex), CD163 mouse monoclonal antibody
(1:100 dilution; Genetex) and prostate specific antigen (PSA) goat polyclonal antibody
(Santa Cruz; 1:100) all prepared in 0.1% Triton-X/PBS. In brief, TMAs were baked for
30 minutes at 60°C prior to deparaffinization/dehydration in a series of (2) 100% xylene,
(1) 100% EtOH, (1) 95% EtOH, and (1) 70% EtOH immersions. Antigens were then
retrieved using Dako PT Link per manufacturer’s instructions. Next, TMAs were warmed
in 30°C oven for 20 minutes in preparation for staining. Using the Dako Autostainer
instrument slides were first blocked with 10% normal goat serum (NGS)/0.4% Triton
X/1X PBS for 20 minutes, followed by incubation with primary antibodies for 1hr at
room temperature. TMAs were then washed and incubated with appropriate secondary
antibodies at a concentration of 1:100 (Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor
26
488 goat anti-rabbit IgG, Alexa Fluor 594 donkey anti-goat IgG and/or Alexa Fluor 594
goat anti-rabbit IgG; Invitrogen) for 1 hour and counterstained with DAPI (1:1000) for 1
minute. To reduce native tissue fluorescence TMA were then incubated with 0.1% Sudan
Black B (SBB)/70% ethanol for 20 minutes at room temperature followed by three, 8
minute washes. Finally coverslips were mounted onto slides with Vectashield mounting
media for fluorescence (H-1000; Vector Laboratories). Analysis of macrophage marker
distribution was carried out using the LSM 700 confocal microscope (Zeiss) at 40x oil
magnification.
Transwell Migration Assay
At roughly 80% confluency, RWPE1, LNCaP, DU145 and PC3 cells were
washed in PBS and serum starved in their respective starvation media (RPMI-1640 or
KSFM) for 48 hours in 37°C and 5% CO2. For control conditions serum-free IMDM,
KSFM and RPMI-1640 medium was also incubated at 37°C in 5% CO2 for 48 hours.
After 48 hour serum starvation, CM was removed from prostate cells and controls and
transferred into labeled 15ml conical tubes on ice. CM was then clarified via
centrifugation at 2000 x g at 4˚C for 15 min. After centrifugation, CM was transferred to
newly labeled 15ml conical tubes and placed on ice. Using 24-well plate/5-µm-pore size
polyvinylpyrrolidone-free polycarbonate transwell filters (Corning), 600µl of clarified
CM was added to the lower chamber of the transwell migration plate, and 5 x 104 SC
peripheral blood monocytes in 100µl were seeded onto polycarbonate filter inserts and
incubated at 37°C/5% CO2. Monocytes were allowed to migrate toward CM for 24 hours,
after which inserts were removed and stained using Hemacolor staining solution (EMD
27
Millipore). Stained (migrated) cells on underside of transwell insert were manually
counted using a light microscope.
Western Blot Analysis
Monocytes, PCa cells or macrophages derived from our experimental conditions
were collected and lysed for 5 minutes in 50µl of 1% Triton X-100, 2mM EGTA, 1mM
phosphatase/protease inhibitor cocktail, and 1X PBS). To ensure complete cell lysis, cell
lysates were then flash frozen/lysed in liquid nitrogen 3 times for 5 minutes each. Lysates
were then centrifuged, protein quantified using Bradford assay, and samples were
prepared containing twenty to forty micrograms of total proteins in loading buffer. Just
before samples were loaded on an SDS–PAGE, they were boiled at 100°C for 5 minutes.
Samples were then separated on SDS-PAGE gel and transferred onto polyvinyl difluoride
(PVDF). The immunoblots were incubated in 3% bovine serum albumin (BSA), 1X TrisBuffered Saline-Tween 20 (TBS-T) for 1 hour at room temperature, incubated with
primary antibodies overnight, followed by incubation with appropriate horseradish
peroxidase (HRP) conjugated secondary antibodies for 1 hour at room temperate.
Immunoblots were developed using enhanced chemiluminescence (ECL) method and
scientific imaging film.
ROS Microplate Assay
Monocytes (1 x 104 cells/well) were plated, differentiated and polarized to
M1macrophages (as previously described) on high-throughput screening (HTS) 96-well
transwell inserts (0.4µM pore size) 3 days prior to experiment. One day prior to
experiment, normal (RWPE1) and cancerous (LNCaP, DU145 and PC3) prostate
28
epithelial cells (2 x104 cells/well) were seeded in the bottom wells of transwell 96-well
black plate and incubated in 37°C/5% CO2 overnight to ensure between 70-80%
confluency on the day of the experiment. Several wells were left empty for the
background fluorescence control measurements. On the following day, supernatant was
removed and prostate cells were carefully washed with 1X Wash Buffer. Transwell
inserts containing M1 macrophages were then inserted into 96 well plate containing
prostate cells and allowed to co-culture for 3 hours. Positive control samples were treated
with ROS Inducer (Pyocyanin) 30 minute before endpoint analysis, and negative control
samples were treatment with the ROS inhibitor, N-acetyl-L-cysteine (NAC), 1hr prior to
experimental end. M1 macrophages and positive/negative controls were removed and
ROS/superoxide detection solution was added (100µl/well) to each well and incubated
for 60 minutes at 37°C in the dark. Finally, plates were read (bottom reading), without
removing the detection mix, using a fluorescence microplate reader and standard
fluorescein (Ex=488nm, Em=520nm) and rhodamine (Ex=550nm, Em=610nm) filter
sets.
Invasion Assay
Prostate epithelial cell invasion was assayed using the BioCoat Matrigel invasion
chambers with 8.0μm PET membrane in 24-well plate format (Corning; 354480). To
prepare plate, 500μl of warm (37°C) bicarbonate based culture medium (IMDM or
RPMI) was added to the inserts and to the bottom wells. Plates were allowed to rehydrate
for 2 hours in humidified tissue culture incubator (37°C; 5% CO2 atmosphere). After
rehydration, media was carefully removed without disturbing the layer of Matrigel matrix
29
on the membrane. PCa cells (5 x 104) were then resuspended in 500μl of serum-free
IMDM (or various experimental conditions being tested). 750μl of desired
chemoattractant (SDF1α + phenol-free/serum free RPMI) or serum-free medium (phenol
free RPMI) was added to the bottom well. Using sterile forceps, empty inserts were
transferred to the wells containing the chemoattractant or serum-free media. Previously
prepared cell suspensions (500ul) was then added to the inserts and incubated for 22hrs in
a humidified tissue culture hood, at 37°C, 5%CO2 atmosphere. To remove non-invading
cells, a cotton tipped swab was used to gently swabbing the membrane surface.
Scrubbing was repeated with a second swab moistened with medium to ensure all noninvaded cells were removed. Prior to staining cells, they were fixed by adding 500μl of
100% methanol to the appropriate number of wells. Invaded cells were then stained using
(Hemacolor staining solution (EMD millipore) following manufacturer’s instructions.
Finally, inserts were allowed to dry upside, overnight. Chemotaxis was evaluated by
manually counting the cells that migrated to the lower surfaces of the polycarbonate
filters.
Anchorage-Independent Colony Formation Assay
Using the CytoSelect 96-Well Cell Transformation Assay (Soft Agar Colony
Formation) kit (Cell Biolabs; CBA-130), agar Solution (1.2%) and 2X DMEM/20% FBS
mixture was prepared and then added to the wells of a black polystyrene flat bottom 96
well plate (with micro-clear bottom) suitable for fluorescence measurement. The plate
was then transferred to 4°C for 30 minutes to allow the base agar layer to solidify.
Prostate epithelial cells were then harvested and resuspended in complete medium at 4 x
30
105 cells/ml. Equal volumes of 1.2% Agar Solution, 2X DMEM/20% FBS media, and
cell suspension (1:1:1) was mixed and transferred to each well of the 96-well flat-bottom
microplate already containing the solidified base agar layer (25 µL of cell suspension
containing 1,000-10,000 cells/well will be seeded). Plate was then placed in 4°C for 15
minutes to allow the cell agar layer to solidify. After cell agar layer solidified, co-culture
conditioned medium (CM) or control CM was added to each well and the plate was
incubated 6-10 days at 37°C and 5% CO2. Cell colony formation was monitored and
examined under a light microscope. Finally, culture medium was removed and agar
solubilization solution was added to each well of the 96-well plate. Plate was incubated
for 1 hr at 37°C. Cells were then lysed and the plate was incubated at room temperature
for 15 minutes. Cell lysate (10 µL) was then transferred to another black polystyrene flat
bottom 96 well plate (with micro-clear bottom) suitable for fluorescence measurement
and CyQuant solution was added to each well. Plates were incubated for 10 minutes at
room temperature and read using a microplate reader (fluorescence 485/520 nm filter set)
Statistical Analysis
Data are presented as the mean ±SE of at least three independent experiments.
The data were analyzed for two-way ANOVA or Student t -test. All statistical analyses
were done, and all graphs were generated using GraphPad Prism 5.0 software
(GraphPad). P values less than 0.05 were considered significant.
31
Figure 7: Cell Transformation Assay Principle. Reprinted from Cell Biolabs, Inc. Retrieved October 28,
2015,
from
http://www.cellbiolabs.com/sites/default/files/CBA-130-cell-transformation-assay.pdf.
Copyright © 2004-2012: Cell Biolabs, Inc.
CHAPTER IV
RESULTS
iNOS detection positively correlated with human prostate cancer tissue samples
To determine whether inflammatory macrophages are associated with prostate
malignancies, we conducted immunohistochemical (IHC) and immunofluorescent (IF)
analysis of human prostate tissue microarrays (TMAs). Tissues representative of normal,
benign hyperplasia, chronic inflammation, and a range of malignant prostatic lesions
were analyzed for distribution of the M1 macrophage marker, inducible nitric oxide
synthase (iNOS or NOS2) and cluster of differentiation 163 (CD163; M2 marker). As
indicated in Figure 8, brown (positive) staining, iNOS was detected in malignant tissues
where positive iNOS detection was more prominent in the earlier stage of malignancy
(Figure 8B-C). iNOS was also present in advance disease specimens, but to a lesser
extent. As expected, there was little to no iNOS detected in normal adjacent tissues
(Figure 8A). Confocal images of tissues stained with immunofluorescent (IF) iNOS (red)
and CD163 (green) reveal a similar pattern (Figure 9). iNOS-positive macrophages were
detected in BPH, chronic inflammation, and localized prostate cancer samples. CD163
detection, on the other hand, was more abundant in the advance metastatic tissue
specimens. Again, little to no detection of M1 or M2 markers was observed in normal
prostate tissues (Figure 9).
32
33
Figure 8: IHC analysis detected iNOS in human prostate tissue microarrays. A) Normal adjacent
tissue. B) Stage II prostate carcinoma. C) Stage IV prostate carcinoma. Positive iNOS expression is
indicated by brown staining. Hematoxylin (blue) was used as a counterstain. Scale bar=20μm. Arrows
indicate positive iNOS staining.
Figure 9: IF analysis of iNOS expression in prostate cancer tissue. IF analysis detected M1 and M2
macrophage expression in human prostate tissue microarrays. Confocal images of IF staining of iNOS
(red) CD163 (green). Nuclei are revealed by DAPI staining (blue). 20X magnification.
34
Prostate cancer cells recruited human monocytes
LNCaP, DU145 and PC3 cells, each exhibiting varying degrees of aggressiveness,
along with normal prostate epithelial cells (RWPE-1) were utilized to model the in vivo
interaction between prostate epithelial cells and macrophages at different stages of
cancer. RWPE-1 cells were derived from the peripheral zone of a histologically normal
adult human prostate, while human LNCaP cells originated from supraclavicular lymph
node metastasis and are poorly aggressive. DU145 cells, obtained from brain metastasis
of a male with grade II prostate cancer, exhibit moderate metastatic potential. The highly
metastatic line, PC3, was isolated from metastases in the lumbar vertebra of a patient
with grade IV prostatic adenocarcinoma and is useful in investigating the biochemical
changes in advanced, malignant prostate cancer (Table 1).
Table 1: Human Prostate Cancer Cell Lines
Using this array of prostate epithelial cells, we assessed the ability of PCa cell
lines of vary aggressiveness, to recruit macrophage precursors (monocytes), via a
transwell migration assay. As detailed in the methodology section, conditioned media
(CM) collected from each prostate cell line was used as chemo-attractants in the bottom
35
chamber, in which human monocytes (SC) seeded in the upper chamber of the transwell
insert, were allowed to migrate towards. We observed that CM from LNCaP, DU145 and
PC3 cells were able to efficiently recruit circulating monocytes, as demonstrated by their
ability to migrate towards CM in the bottom chamber (Figure 10). Monocytes seeded in
control, unconditioned media (IMDM or RPMI) demonstrated insignificant migration.
Likewise, CM from RWPE-1 failed to induce significant chemotaxis of monocytes
(Figure 10).
Figure 10: PCa cells recruited human monocytes. Monocytes were serum starved and allowed to
migrate for 24h toward CM from RWPE-1, LNCaP, DU145 and PC3 cells. Negative control conditions
included: IMDM (monocyte culturing media), KSFM (RWPE1 culture media) and RPMI (LNCaP, DU145
and PC3 culturing media). Exposure to the chemokine SDF1α was established as a positive control.
Graphical representation of results also included.
36
Classical stimuli-mediated monocyte differentiation and polarization
For the classical induction of monocytes to a terminal macrophage differentiation
state, SC cells were cultured in the presence of 97 nM (60ng/ml) phorbol myristate
acetate (PMA) for 6 h, followed by treatment with interferon gamma (IFN-γ) and
lipopolysaccharide (LPS) or IL-4, generating M1 and M2 macrophages, respectively.
After differentiation/polarization, the media was removed and replaced with complete
media for 24 h. As shown in Figure 11, monocytes cultured 72 hours with PMA and/or
M1/M2 components caused the once non-adherent monocytes to adhere to the culture
dish and displayed evident morphological changes (filopodial extensions), characteristic
of mature macrophages (Figure 11). Subsequent to classical differentiation, SC cells were
collected and analyzed via immunoblot analysis. As expected, SCs treated with IFNγ and
LPS had higher expression of the M1 marker, COX2. Cells treated with IL-4 displayed a
higher expression of COX1, a well-established marker of M2 differentiation. Results
from this experiment confirmed our method of M1 and M2 differentiation was effective
and therefore subsequent experiments conducted beyond this point involving
differentiated macrophages were done so using these experimental parameters.
Macrophage co-culture drives SC differentiation
In order to analyze if exposure to tumor microenvironment features can affect
monocyte differentiation, we incubated human peripheral blood monocytes with normal
(RWPE1) and cancerous prostate epithelial cells (LNCaP, DU145, PC3) of varying
aggressiveness. The results show that LNCaP, DU145 and PC3’s, but not RWPE1s, are
able to induce evident morphological changes of monocytes that became larger, with
37
Figure 11: Classical monocyte to macrophage differentiation. A) Phase-contrast microscopy
demonstrating morphological changes in monocytes in response to PMA (60ng/ml) and subsequent M1 or
M2 polarization with LPS (0.1μg/ml) and IFNγ (20ng/ml) or IL-4 (20ng/ml), respectively. B) Protein
expression of M1 markers (iNOS and COX-2) and M2 markers (COX-1 and TNFα) assessed via western
blot analysis.
ruffling membrane typical of macrophages (Figure 12A). In order to analyze the
phenotype of the co-culture differentiated macrophages, we assessed the expression of
inducible nitric oxide (iNOS) and COX-1. iNOS was highly expressed by macrophages
cultured with the lowly aggressive cell line, LNCaP. In contrast, macrophages cocultured with the moderately aggressive, DU145 and highly metastatic PC3 cells
displayed little to iNOS expression. Instead, COX-1, an M2 marker, was highly
expressed in DU145 and PC3 co- cultured monocytes (Figure 12B). Normal, RWPE1
cells were unable to induce morphological or phenotypic changes in monocytes.
38
Figure 12: Prostate cancer cell/monocyte co-culture drives macrophage differentiation. A) Brightfield microscopy demonstrating morphological changes of monocytes in response to co-culture with
normal prostate cells (RWPE-1) and prostate cancer cell lines of varying aggressiveness ( LNCaP,
DU145, and PC3). B) Western blot analysis of co-cultured SC cells probing for M1 and M 2 markers
iNOS (M1) and COX1 (M2).
Exposure to M1s increases ROS in normal and cancerous prostate models
To determine whether co-culture with inflammatory macrophages (M1) increases
oxidative stress in normal (RWPE1) and malignant prostate epithelial cells (LNCaP,
DU145 and PC3), we performed ROS microplate assays. In doing so, cells were stained
with the cellular ROS indicator, c-H2DCF-DA, which upon oxidation by ROS forms the
highly fluorescent compound, 2’, 7’ –dichlorofluorescein (DCF), which can be detected
by fluorescence spectroscopy. Normal and cancerous cell lines were incubated with cH2DCF-DA in phenol-free RPMI or phenol-free RPMI 1640 alone (control) for 20
39
min at 37°C. Cells were then mono- or co-cultured with M1 macrophage CM for 1, 3,
and 6 h. Fluorescence, in response to intracellular oxidation of c-H2DCF-DA, was
measured with a microplate reader at 485nm (excitation) and 535nm (emission)
wavelengths per manufacturer's instructions. As shown in Figure 13, compared to monocultured cells, M1 co-cultured cells, exhibited significantly greater fluorescence
indicating higher oxidation levels. This trend was observed across all cell lines
suggesting exposure to M1 macrophages alters the redox state of prostate cells (Figure
13).
Figure 13: Exposure to inflammatory macrophages increases ROS in PCa cells. Prostate epithelial
cells were either mono or co-cultured with M1 macrophages for the 3h. Cells were then labeled with
10µM cH2DCF-DA for 60 min at 37°C. Fluorescence was measured using a microplate reader
(Excitation:488nm /Emission:520 nm). * p<0.05. PYO-Pyocyanin (ROS inducer); APO-Apocynin (NOX
inhibitor).
M1 macrophages promote prostate tumorigenesis in vitro
To confirm that RWPE-1 cells were indeed undergoing a tumorigenic process
after co-culture, RWPE-1 cells in M1 conditioned media, were plated on soft-agar to
determine their anchorage independent growth. As expected, non-tumorigenic RWPE1cells were
40
able to form colonies on soft-agar after co-culture with M1 macrophage conditioned
medium (Figure 14).
Figure 14: M1 macrophages promote PCa cell anchorage-independent growth. Colony formation in
soft agar assay of RWPE-1 cells (alone) and RWPE-1 cells co-cultured with THP-1cells. A bar graph,
representative of six randomly chosen fields is shown.
To evaluate the effects of exposure to inflammatory macrophages in prostate
epithelial cells, we analyzed cell invasion. We found that post-M1 conditioned media
exposure, all cell lines assay demonstrated an increased in invasiveness through the
basement membrane-like material, matrigel. Interestingly, M1 CM exposure elicited a
strong invasive spur in LNCaP cells; second only to PC3’s which are inherently highly
invasive (Figure 15).
41
RWPE1
LNCaP
DU145
PC3
Figure 15: M1 macrophages increase invasiveness in PCa cells. Monocytes were differentiated for 3
days and then serum starved for 48 h to obtain CM. PCa cells were incubated with collected CM for 24
h, (or serum free medium as a control), and then were allowed to invade toward medium containing
SDF1α, serving as chemoattractant, for an additional 24 h. Invading cells were counted and a bar graph,
representative of six randomly chosen fields, is shown.
CHAPTER V
DISCUSSION
In recent years, evidence has mounted supporting the idea that inflammation
within the normal adult prostate is not uncommon.26 In most cases, the cause of prostatic
inflammation is unclear; however, various potential causative factors exist, including
direct bacterial and viral infection, urine reflux, dietary factors, and hormonal changes.26
More recently, this area of research has gained more attention as researchers continue to
investigate a potential link between chronic prostatitis and prostate cancer. For example,
in 2010, an epidemiological study found an increased risk for prostate cancer in men with
a history of prostatitis.45 Another study revealed that recurrent E. coli infections in the
murine prostate induced the formation of PIN lesions, due to the persistent secretion of
free radicals and inflammatory cytokines by infiltrating leukocytes.49 Interestingly,
among the leukocytic infiltrates, macrophages are generally regarded as the key players
supporting cancer progression within the tumor milieu.26
It is generally recognized within the scientific community that tumor-associated
macrophages display an M2 phenotype, promoting pro-tumoral functions such as cell
survival, proliferation, and angiogenesis. These functions are regulated through
production of important mitogens, growth factors and enzymes.76 M1 macrophage
42
43
activation, on the other hand, is characterized by a high capacity to present antigen
type I response, and increased production of toxic intermediates (nitric oxide (NO) and
reactive oxygen species. Based on this, M1 macrophages are generally considered to be
potent effector cells that kill micro-organisms and tumor cells, and produce copious
amounts of pro-inflammatory cytokines.76 In general, high levels of TAMs are often,
although not always, correlated with a bad prognosis, and recent studies have also
highlighted a link between their abundance and the process of metastasis.77
Increasing studies have also shown that TAMs can either enhance or antagonize
the antitumor efficacy of cytotoxic chemotherapy, cancer-cell targeting antibodies, and
immunotherapeutic agents. Therefore, their role in mediating resistance to cancer
therapies makes them an attractive target in drug development. A number of studies have
demonstrated that through secretion of various chemo-protective factors such as MMP-9
and cathepsins, TAMs are able to reduce chemotherapy-induced antitumor responses,
thereby promoting cancer.78 Overwhelmingly, the models used in such TAM-related
studies are reported to possess characteristics of M2-like macrophages with protumor and
immunosuppressive activity already prevalent in the tumor microenvironment.79 It should
also be noted that the predominant detection of M2 macrophages reflects the late stage of
tumor progression, while the presence, type and role of macrophages in lower staged
neoplasias is only marginally studied. This observation raises the question of: (i) whether
the preexisting proinflammatory environment in glandular tissues such as the prostate,
before tumorigenesis, would favor M1 or M2 polarization of macrophages; and (ii) the
role of such macrophages in initiating malignant transformation.
44
In the present study, we sought to explore this question and believe to have
discovered a potentially novel function of M1 macrophages in promoting a pro-tumor
microenvironment whereby oxidative stress and inflammation drive malignant
transformation of the prostate. It is important to note, that our observations are not
ignorant of the firm role of M2s in fueling cancer, but instead, aim to compliment the
well-established complexity of macrophages and the tumor microenvironment as a
whole.
In contrast to the preeminent paradigm regarding macrophage infiltration in solid
tumors, herein we demonstrate the involvement of M1 macrophages in tumor initiation,
and to a lesser extent, tumor progression. In support of this notion, many studies have
reported the role of inflammation and oxidative stress in promoting disease. Likewise, the
ROS-generating activity of M1 macrophages is well-established. Therefore, it is
conceivable that M1s may inadvertently drive cancer progression. While this concept is
still only emerging, a few studies have made small inferences. Sebens et al. observed that
exposure of normal colon epithelial cells to inflammatory macrophages induced activity
of the anti-oxidative transcription factor, Nrf2, resulting in proteasome activation and
protection from apoptosis. 13 Similarly, Comito et al. inadvertently demonstrated that M1
macrophages, although to a lesser extent than their M2-polarized counterparts, increased
invasiveness of PCa cells and transformed normal human prostate fibroblasts to tumorassociated fibroblasts. 14 Fascinatingly, Fang et al. recently reported that persistent coculturing of immortalized normal prostate epithelial cells with macrophages, in the
absence of carcinogens, induced prostate tumorigenesis.15 Our findings underscore the
45
potential role of M1 macrophages in prostate cancer with regard to their production of
ROS.
CHAPTER VI
CONCLUSION
TAMs are key orchestrators of the smoldering inflammation present in the tumor
microenvironment. In the majority of experimental and clinical studies, TAMs have been
associated with cancer progression. Their production of various growth factors for
epithelial and endothelial cells, as well as inflammatory cytokines and chemokines
contribute to tumor survival, proliferation and invasion. In addition, immunosuppressive
mediators released by local inflammatory or tumor cells extinguish host-mediated antitumor responses and facilitate tumor progression. Thus TAMs appear as attractive
candidate of novel therapeutic strategies. Three major aspects of TAM, potentially
amenable of therapeutic interventions are: (i) inhibition of their recruitment and/or of
their survival at the tumor site; (ii) inhibition of their positive effects on angiogenesis and
tissue remodeling; (iii) reversal of their immune-suppression and restoration of antitumor cytotoxicity.
While an increasing amount of pre-clinical studies specifically targeting TAM
have yielded encouraging result, these results have not yet been successfully translated
into the clinic. Furthermore, the role of M1 macrophages in fueling cancer has not been
exploited therapeutically. Therefore, continued characterization of tumor associated
macrophages represents a valuable strategy to complement established anticancer
treatments.
46
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