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Studying cancer and metastasis: how multiphoton
microscopy contribute to this
Christa Eekhuis, University of Utrecht
Abstract
Cancer is worldwide a deadly disease and results in many deaths. Tumor formation can be
induced by several factors, such as mutations in genes and viruses. Genes affected by
mutations are mainly tumor suppressor genes and oncogenes, which results in the up- or
downregulation of cellular processes. During tumor formation metastasis can occur as
well and this result in many deaths due to cancer. Metastasis is a complex and dynamic
process, consisting of many steps, like cell detachment and intravasation.
Immunohistochemistry is mainly used to study metastasis, but nowadays intravital
microscopy techniques are used as well. With intravital microscopy we are able to answer
the questions, which still remain about metastasis. We are now able to follow metastasis in
vivo over time with intravital microscopy. Multiphoton microscopy is one of the
techniques which is used to study metastasis. With the use of fluorescent proteins, such as
GFP and photoswitchable proteins, it is possible to follow single cells over time during
metastasis to gain more knowledge about metastasis. This can help in developing
treatments for cancer, thereby preventing the formation of secondary and tertiary tumors.
Introduction
Cancer
Cancer is worldwide a deadly disease and results in many deaths. Almost 90% of
the deaths are caused by metastasis of tumors(Gupta & Massague, 2006). Cancer
development is a process in which cellular processes are altered, like cell
proliferation and apoptosis. Most of the time mutations occur in genes involved
in these processes, resulting in the up- or downregulation of genes. The genes
mutated are mainly oncogenes or tumor suppressor genes(Wrzeszczynski et al.,
2011). A variety of factors can cause the formation of tumors. One of these causes
are viruses, like the human papillomavirus (HPV), which can cause cervix
cancer(Faridi, Zahra, Khan, & Idrees, 2011).
Second, mutations in the germline can occur as well, resulting in
hereditary diseases. The most studied hereditary diseases are breast cancer
(BRCA1/2 mutations) and HNPPC (APC, β-catenin, K-ras mutations)(Jass, 2004;
Shulman, 2010) BRCA1/2 are tumor suppressor genes, which are involved in
DNA repair, but when mutated they can cause breast cancer, but ovarian cancer
too(Shulman, 2010). Hereditary non-polyposis colorectal cancer (HNPPC) is
responsible for a small percentage of colorectal cancer. Females with this
syndrome can develop cervix and ovarian cancer as well(Jass, 2004). Beside
BRCA1/2 and HNPPC, other syndromes, like VHL, can cause cancer too(Jass,
2004; Moore et al., 2011; Shulman, 2010).
Most tumors arise from somatic mutations caused by internal and
external factors(Wrzeszczynski et al., 2011). External factors, like UV and
chemicals, and internal factors, like mitochondria and peroxisomes, induce the
formation of reactive oxygen species (ROS) in your body(Valko et al., 2007; Veal,
Day, & Morgan, 2007). Most ROS will be scavenged by antioxidants, but when the
ROS levels are too high to be scavenged, it will cause damage to DNA, lipids and
proteins. In addition, ROS can induce chromosomal instability as well. The DNA
damage can result in gene mutations or altered gene expression, which
contributes to tumor formation(Valko et al., 2007). Furthermore, chromosomal
rearrangements, like translocations and deletions, and epigenetic alterations,
like DNA methylation, contribute to tumor formation too(Wrzeszczynski et al.,
2011).
Metastasis of tumors
One of the hallmarks of cancer is metastasis (figure 1) and this is the cause of
most deaths due to cancer. Metastasis is a very dynamic process and during
metastasis cancer cells spread throughout the whole body via blood vessels,
lymphatic vessels and serosal surfaces, before new colonies can be formed in
other organs(Bacac & Stamenkovic, 2008; Hanahan & Weinberg, 2011). Most
tumors spread throughout the body via the lymph nodes and blood vessels. Only
a small group of tumors, like ovarian cancer, has the ability to metastasis
transcoelomic(Bacac & Stamenkovic, 2008).
Before metastasis can occur, cancer cells have to overcome several
barriers in your body. Therefore metastasis can be seen as an evolutionary
process, because evading barriers results in selection of cancer cells, which are
able to survive during this process(Gupta & Massague, 2006). Metastasis is a
dynamic process, which can be divided into two phases: the translocation of
cancer cells to other organs (1) and the ability of these cells to adapt to the new
environment and develop a secondary or tertiary tumor (2)(Bacac &
Stamenkovic, 2008; Chaffer & Weinberg, 2011). These two phases consists of
many steps, which are essential for the formation of secondary or even tertiary
tumors.
Figure 1| The different steps
of metastasis. First there is
detachment of tumor cells from
the tumor, resulting in breaking
through the basal membrane
into the surrounding tissue
stroma (invasion). After
invasion, the cells migrate
towards collagen fibers towards
the blood and/or lymphatic
vessels and intravasation
occurs. Tumor cells move via
the circulation to other organs,
where they extravasat and start
proliferating, inducing
angiogenesis en form a
secondary or tertiary
tumor(Bacac & Stamenkovic,
2008).
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Cell detachment
The first step of metastasis, is the loss of cell adhesion from the tumor(Bacac &
Stamenkovic, 2008; Gupta & Massague, 2006). Most tumors consist of epithelial
cells, which are strongly zipped to each other, via membranes like tight junctions
and desmosomes(Garber, 2008; Thiery & Sleeman, 2006). Epithelial cells
polarize apical-basolateral and are only able to move within the epithelial
layer(Thiery & Sleeman, 2006). If epithelial cells want to metastasize from the
tumor throughout the body they have to undergo a transformation into
mesenchymal cells. This is called the epithelial-to-mesenchymal transition
(EMT)(Garber, 2008; Mani et al., 2008; Thiery & Sleeman, 2006). When EMT
occurs, the epithelial cell loses most of its characteristics and transform into a
mesenchymal cell (figure 2). The transformed cell also acquire characteristics
from a mesenchymal cell, resulting in the change of the architecture of the cell
and its behavior(Garber, 2008; Thiery & Sleeman, 2006). The architecture of
mesenchymal cells look like fibroblasts, they are not tightly zipped to each other
and these cells are more motile then epithelial cells(Thiery & Sleeman, 2006).
Figure 2| The plasticity cycle of the epithelial cell. It shows which events occur during the
transformation of epithelial cells into mesenchymal cells and vice versa. EMT and MET are
induced and regulated by different effectors, such as growth factors and adhesion. The MET and
EMT effectors can inhibit each other. Furthermore, important processes, like the formation of
tight junctions during MET, are indicated. During EMT and MET, several markers have been
found, which are typical for mesenchymal and epithelial cells(Thiery & Sleeman, 2006).
EMT is normally an important key player in embryogenesis(Garber, 2008; Mani
et al., 2008; Thiery & Sleeman, 2006), because without EMT development stops
at the blastula stage and further development cannot occur anymore. During
embryogenesis, EMT is involved in several processes, like invagination and
multi-layering. Due to EMT the three layered embryo can be formed, but also
other structures, like vertebrae and craniofacial structures. Furthermore, EMT
mediates the loss of the mullerian duct in males. Extracellular signals can induce
EMT, during embryogenesis (figure 3)(Thiery & Sleeman, 2006). Mesenchymal to
Page | 3
epithelial transition (MET) occurs during embyrogenesis as well, especially
during the development of kidneys(Mani et al., 2008), the formation of coelomiccavity and during somitogenesis(Thiery & Sleeman, 2006).
There are a lot of transcription factors, like Twist and Snail, which are
involved in triggering EMT in cells during embryogensesis. These transcription
factors are found to be involved in processes related to cancer formation and
metastasis, like invasiveness and resistance to apoptosis(Mani et al., 2008). Not
only transcription factors trigger EMT, but also growthfactors, like TGFβ, and
parts of the extracellular matrix (ECM), like collagen(Thiery & Sleeman, 2006).
When EMT is induced, desmosomes, β-catenin and E-cadherin are repressed,
which results in the loss of cell-cell adhesion and changes in the cytoskeleton
(Buda & Pignatelli, 2011; Mani et al., 2008). Other components are
downregulated as well, especially those who are involved in cell-cell adhesion
like vinculin and plakoglobin(Thiery & Sleeman, 2006).
Figure 3| Overview of molecular pathways involved in EMT. Several pathways are able to
activate EMT and there is crosstalk between some pathways. When receptor tyrosine kinases
(RTKs) are activated, activation of Rac occurs as well to induce cytoskeleton rearrangement. The
TGFβ pathway activates a lot of players, from which Twist and Snail are the proteins which
induce EMT, by repressing E-cadherin or directly influence the EMT programme. Furthermore,
MMPs are able to induce EMT too, but it is not known which receptors are involved(Thiery &
Sleeman, 2006).
E-cadherin is a member of the cadherin family, which are transmembrane
glycoproteins involved in cell-cell adhesion. One of the groups of the cadherin
family is the classical type I cadherins. The classical type I cadherins consist of
five cadherins, including E-cadherin and N-cadherin(Van den Bossche, Malissen,
Mantovani, De Baetselier, & Van Ginderachter, 2011). The cytoplasmic region of
Page | 4
the E-cadherin is bound to β-catenin, which is essential for cadherin-based cellcell adhesions, and p120 and this complex binds to the actin cytoskeleton. The
extracellular part of E-cadherin binds to another E-cadherin to form dimers,
resulting in cell-cell adhesion(Van den Bossche et al., 2011; Yip & Seow, 2011).
When EMT occurs in tumors, E-cadherin is downregulated by transcription
factors like Snail and Twist, but also by hypermethylation of the promoter. Due
to the loss or downregulation of E-cadherin and β-catenin cell detachment can
occur, which can result in metastasis(Yip & Seow, 2011).
The down regulation of E-cadherin and β-catenin, results in the up
regulation of other proteins, like N-cadherin and vimentin(Mani et al., 2008). Ncadherin is an important player in EMT and is present in neural tissue, where it
is highly expressed and localizes at adherens junctions. It plays a role in cell-cell
and cell-matrix contacts and the cytoplasmic part is involved in signaling
pathways. In many tumors E-cadherin is decreased, while N-cadherin is
increased, probably playing an important role in cell motility. Studies have
shown that N-cadherin can promote metastasis and invasiveness of tumor cells
in different cancer models(Jennbacken et al., 2010). Beside N-cadherin other
proteins are up regulated, which are characteristic for mesenchymal like
cells(Mani et al., 2008).
Although a lot of the researchers are convinced that EMT is involved in
metastasis, there are still some researchers which are sceptic about this. Studies
have shown that Snail and Twist are highly expressed in human tumors, which
are highly invasive. However, pathologist David Tarin, MD. PhD. FRCPath is
sceptic about EMT. He thinks that EMT cannot occur in tumors, because it is
never detected in tumor samples, it is not shown in animal models for cancer, the
markers used are not specific enough for mesenchymal cells and due to the
genetic instability of tumors these markers are unreliable. Taken this together,
there must be a change in epithelial cells to become more motile and invasive,
but it is not shown yet that this occurs due to a total EMT(Garber, 2008).
Invasion of the tumor cell
When the cancer cell is detached from the tumor mass, the tumor cell can invade
the surrounding tissue stroma or the blood vessels within the tumor(Thiery &
Sleeman, 2006). Lymphatic vessels are not found in tumors, so to metastasize via
the lymphatic system, tumor cells have to invade the underlying stroma(Pepper,
Tille, Nisato, & Skobe, 2003). If a tumor wants to grow, angiogenesis is essential
and this is mainly driven by the growthfactor VEGF(Amin et al., 2011), but also
by other factors like matrix metalloproteinases (MMPs), which remodeling the
basement membrane of the blood vessels. These growthfactors and proteases
are expressed and secreted by tumor cells(Kalluri, 2003). When VEGF binds to
its receptor, this induces chemotaxis and mitogenesis of endothelial cells and the
permeability of vessels increases(Zhang et al., 2011). The permeability of vessels
increases, so new vessels can sprout from these excisting vessels. Tumor vessels
show already morphological abnormalities, like the lack of a monolayer of
endothelial cells on the blood vessels. Furthermore, the cells lose focal
intercellular gaps and interconnections, probably resulting in leaky vessels
within the tumor. Due to the leakiness of the vessels, tumor cells are able to
Page | 5
invade the blood vessels within the tumor to spread throughout the
body(McDonald & Baluk, 2002).
Beside invading blood vessels, tumor cells can invade the surrounding
tissue stroma to metastasis. Matrix metalloproteinases (MMPs) are key players
in invading the tissue stroma by tumor cells. MMPs are involved in biological
processes, like migration(), tissue remodeling(Kessenbrock, Plaks, & Werb,
2010), angiogenesis(Kalluri, 2003) and the degradation and cleavage of ECM
proteins(Egeblad & Werb, 2002). One of the proteins cleaved by MMPs is Ecadherin and this results in more invasive cells(Egeblad & Werb, 2002). In
tumors, carcinoma and stromal cells secrete and express MMPs, resulting in the
degradation of basement membrane (BM) components(Al-Raawi, Abu-El-Zahab,
El-Shinawi, & Mohamed, 2011). The BM is a ECM, which is specialized, because it
forms a barrier between the epithelial cells and the underlying stroma. Signals
are transmitted in the ECM via cell-surface adhesion receptors and the ECM
maintains cell-cell adhesion. The ECM contains a lot of molecules, including
collagens, glycoproteins and proteoglycans(Egeblad & Werb, 2002). The BM is
mainly made of networks of type IV collagen and laminin, which are interwoven
and form a platform. Furthermore, the BM has pores within the network of type
IV collagen, with a size of 50nm. Normally cells cannot migrate through pores
which are smaller than 2,0µm,
so the BM probably acts as a
physical barrier to prevent
cells from invasion into the
underlying stroma(Rowe &
Weiss, 2009). Breakdown of
the basement membrane
makes invasion possible for
carcinoma cells, within the
underlying stroma, blood
vessels and lymphatic vessels.
However, it is not known if
invasion of tumor cells, in the
underlying stroma, relies on
the breakdown of the BM by
MMPs(Al-Raawi et al., 2011).
Figure 4| Multiphoton microscopy to visualize the migration of tumor cells. (a) The use of
multiphoton microscopy resulted in imaging the ECM (purple) and tumor cells (green) at low
magnification. Cells are moving along the ECM away from the tumor by invading the surrounding
tissue. (b) A higher magnification shows that the tumor cells are slightly attached to the collagen
fibers to move towards blood vessels and so on. This picture is a result of the generation of SHG
signals(Condeelis & Segall, 2003).
When the tumor cells have reached the underlying stroma, cell migration occurs.
Cancer cells can move as groups in the form of a chain or as single cells, resulting
in an amoeboid movement or extension-adhesion-retraction movement, like
mesenchymal cell movement(Thiery & Sleeman, 2006). Invasion of groups tumor
cells, occurs in epithelial tumors, like breast cancer, and there are three
hallmarks which characterize this. To migrate in groups of cells, the cell-cell
Page | 6
junctions within the group have to be intact during migration(1), remodeling
occurs of the ECM and the BM has to be rearranged(2). And last, there must be
polarity and the cytoskeleton must be activated to generate the traction force,
resulting in cell movement. To move, the leading cells express integrins,
necessary for the production of adhesion complexes. These adhesion complexes
can connect to fibronectin and collagen and cell movement occurs. Collective cell
movement has advantages, such as the production of promigratory factors in
high concentrations and the inner cells are protected against external factors
during the migration(Friedl & Wolf, 2003).
Single cell movement can occur as well and the invasion of mesenchymal
cells excist out of five steps. These steps include protrusion of pseudopods, focal
contact development, proteolysis, contraction of actomyosin and the trailing
edge have to detach. Mesenchymal cells derived from epithelial cells, due to EMT,
are partially polarized, because rearrangement of F-actin occurs. The focal
adhesions of the cell attach the ECM and the tail of the cell moves due to the
retraction fibers, which contract. TGF-β is a keyplayer in the dedifferentiation of
epithelial cells into mesenchymal cells, resulting in an increased invasion. Beside
mesenchymal cell migration there is amoeboid cell movement. Amoeboid cells,
compared to mesenchymal cells, do not have cell polarity, chemotaxis cannot
occur anymore and the attachment to the ECM is loose. These cells move faster
than mesenchymal cells and collective cancer cells. Furthermore, amoeboid cells
migration is protease independent and does not result in the breakdown of ECM
components, like fibrils. Migration occurs by displacing the fibrils with the use of
mechanical forces, which are actomyosin based. However, this can only take
place when there is no stabilization of the formed pores in the collagen network
and thus no ECM stiffness occurs. Normally there will be protease activity of
MMP14 in collagen networks, which provide amoeboid cell movement, because
the pores within the collagen network are too small to move through. Amoeboid
cell movement is probably a consequence of the use of protease inhibitors or
integrin blocking drugs, which are used to treat cancer. Clinical trials have shown
that MMP inhibitors induce cancer progression via amoeboid cells instead of
preventing it. Moreover, in vitro studies about amoeboid cell invasion were done
under conditions not equal to the in vivo collagen composition and this raises the
debate if amoeboid cell movement is protease indepent(van Zijl, Krupitza, &
Mikulits, 2011).
Intravasation, circulation and extravasation
When the tumor cells are in the underlying stroma, intravasation of blood vessels
and lymphatic vessels can take place. Normally the lymphatic network collects
immune cells and macromolecules from extravasated fluid, before sending them
back to the blood system. If tumor cells want to enter the lymphatic system, they
have to move through the wall of a lymphatic vessel. Tumor cells probably
migrate through the wall of lymphatic capillairies. The wall of these capillairies
lacks pericytes and a basement membrane and the junctions between the
endothelial cells are more sparse, than the junctions in blood vessels. This could
explain why it is easier for tumor cells to enter the lymphatic vessels, but this is
not proved yet and it is still unknown if these junctions make it possible for
tumor cells to enter. When the tumor cell has entered the lymphatic vessel it can
Page | 7
stay there to grow or return to the blood circulation via the thoracic duct(Pepper
et al., 2003).
As mentioned above, MMPs are involved in angiogenesis and are able to
remodel the basement membrane of blood vessels, so tumor cells can enter the
blood circulation. With the use of the chick CAM model, researchers have shown
that MMP9 is involved in intravasation of tumor cells. The blood vessels which
are mainly intravasated by tumor cells, are probably angiogenic blood vessels
instead of mature blood vessels. Intravasation of tumor induced angiogenic
blood vessels versus mature blood vessels is shown with specialized mouse
models. Although MMPs are involved in intravasation of tumor cells in animal
models, this has not been shown yet in cancer patients, raising the question if
MMPs are involved in metastasis in humans(Deryugina & Quigley, 2006).
When the tumor cell enters the lymphatic circulation, it does not have to
overcome barriers, so this could promote survival and dissemination of these
cells. Most studies about metastasis focused on the behavior of tumor cells in the
blood circulation, but not how tumor cells behave in the lymphatic circulation.
Perhaps tumor cells have to overcome certain barriers when entering the
lymphatic circulation, but this still has to be studied(Pepper et al., 2003).
However, when tumor cells enter the blood circulation they have to survive to be
able to form secondary tumors. One way to try to survive is to interact with
blood platelets. Blood platelets facilitate migration and invasion of tumor cells
and the arrest in blood vessels. Tumor cells are able to mimic receptor
expression from blood platelets, resulting in the expression of the GPIIb/IIIa
receptor. Tumor cells and blood platelets interact via bridging proteins, like
fibrinogen, which can bind the GPIIb/IIIa receptor and this protect tumor cells.
Tumor cells use the blood platelets as shields to protect them against phsycial
stressors in the blood circulation and to evade the effector cells of the immune
system(Bambace & Holmes, 2011).
Tumor cells go with the flow of the blood stream, before they arrest in
capillaries(Chambers, Groom, & MacDonald, 2002). When tumor cells arrest,
which is facilitated by blood platelets, they can stay and grow or extravasate out
of the capillary into the surrounding tissue(Bambace & Holmes, 2011; Chambers
et al., 2002). There are several factors involved in extravasation, such as blood
platelets. Blood platelets can help tumor cells to extravasate into the underlying
tissue, because activated platelets can release proteolytic enzymes, like
heparanase and MMPs, which can remodel the BM of blood vessels. In addition,
blood platelets can also activate other proteinases and/or stimulate the
activation of proteinases by endothelial and tumor cells(Bambace & Holmes,
2011). When tumor cells enter the underlying stroma, it is important to form a
new tumor. To develop a new tumor, there must be growth of the cells and to
induce this an reciprocal signaling network will be made by tumor cells. These
signaling networks activate stromal cells to grow, resulting in a secondary
tumor(Langley & Fidler, 2011). Furthermore, angiogenesis has to occur to
sustain the growth of the tumor(Chambers et al., 2002).
Another possibility is that the tumor cells enter the dormant state. This
can occur in both the primary and secondary tumor. Dormancy has advantages
and disadvantages. The disadvantages are that these tumors are too small to be
detected and when the escape the dormant state they grow fast and are most of
the time lethal. However, an advantage can be that tumor progression is paused
Page | 8
and this can take years. When tumor cells are in the dormant state, they are
asymptomatic and occult for a period. Although there is not much known about
tumor dormancy, there are some mechanisms, which are probably involved in
this process. One of these is impaired angiogenesis is associated with dormancy.
In different experimental models tumor cells could not grow, because of
impaired angiogenesis, resulting in asymptomatic small lesions. To escape the
dormant state only one angiogenic factor is enough, which probably affects the
angiogenic balance by inducing a shift. Other mechanisms which are most likely
involved in dormancy are mechanisms such as the ECM, the environment of the
surrounding tissue and the interaction between the normal stromal cells and the
tumor itself. Even though there are many mechanisms, which can induce
dormancy, escaping dormancy is poorly understood(Almog, 2010).
Studying cancer metastasis
It is important to study cancer and especially metastasis, not only to find out how
tumor cells behave in vivo, but also the underlying molecular mechanisms of this
process are important. The knowledge gained about metastasis can be used in
the clinics to develop drugs and the right treatment program for patients. For
many years researchers were only able to study metastasis with techniques, such
as immunohistochemistry, which could only provide static pictures. These static
pictures have advantages such as providing information about the expression of
antigens (e.g. proteins)(Fritschy, 2008) and it quickly generates results(RamosVara, 2005). Nevertheless, there are disadvantages which make it hard to study
metastasis. Metastasis is a highly dynamic process, which can take months and
different time point have to be chosen to study this. At every time point an
animal will be sacrificed to do IHC and due to the diversity within the species, a
lot of animals have to be sacrificed before a valid conclusion can be
made(Ramos-Vara, 2005).
To overcome these problems, tissues of living animals are studied with
microscopes, known as intravital microscopy, which was first done in the 19th
century(Beerling, Ritsma, Vrisekoop, Derksen, & van Rheenen, 2011;
Ntziachristos, 2010). During first years of intravital microscopy, most studies
only focused on the microcirculation and vasculature. Imaging other tissues was
limited, because there was no contrast and the optics available at the moment
were not optimal yet. Later on in the 1950s with the use of an ear chamber, in a
rabbit, metastasis could be visualized. The biggest breakthroughs for intravital
microscopy occurred in the 1990s, when imaging techniques really improved
and animal models were available, which were genetic cancer models and they
could express fluorescent proteins. From that moment on, intravital microscopy
become very important as tool for studies about the underlying mechanisms of
cancer and metastasis(Beerling et al., 2011).
Nowadays, there are several intravital microscopy techniques, which are
used to study cancer and metastasis. One of these techniques is multiphoton
microscopy and in this review I will show why multiphoton microscopy is a good
alternative to study metastasis compared to the other techniques available.
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The advances of Multiphoton microscopy in cancer studies
Immunohistochemistry versus intravital microscopy
In 1941 Coons et al. published a paper describing a new technique, which can
detect cellular antigens with immunofluorescence in different tissues. This was
the beginning of a technique called immunohistochemistry(Ramos-Vara, 2005).
Since the development of IHC, it has become an important tool for both the
research and the diagnostics to study neoplastic and infectious diseases in
animal models. IHC links three disciplines, which are immunology, chemistry and
histology. Aim of IHC is to detect antigens within tissues using specific
antibodies, resulting in the binding between antigen and antibody. This
interaction can be visualized with ultraviolet light, using fluorochromes or with
light microscopy, a histochemical reaction has been performed to color the
antigen-antibody interaction. During the years, IHC became a more complex
technique, because the specificity and sensitivity had to be increased(Mighell,
Hume, & Robinson, 1998; Ramos-Vara, 2005). Nowadays it is possible to observe
one or multiple antigens together or to detect one specific antigen in the same
section of multiple tissues(Ramos-Vara, 2005).
Before IHC can be performed, the tissue has to be fixated. Fixation is
essential for IHC and there are four reasons which support this. First of all, with
fixation cellular components are protected and this includes the structural and
soluble proteins. Secondly, displacement of cell components and autolysis cannot
occur anymore. Third, cellular materials are stabilized and protected against
harmful effects of the following procedures. Last, fixation promote
immunostaining and conventional staining. Although, IHC has advantages such
as the detection of multiple antigens, gaining a lot of information and that
fixation does not affect cellular materials, but there are disadvantages as well. As
mentioned before, metastasis is a process which can take months, so to study
metastasis a lot of time points have to be chosen. For every time point an animal
has to be sacrificed and because there is diversity between the species, even
more animals have to be sacrificed before a statistically valid conclusion can be
made. In addition, cross reaction of antibodies between the species can occur
during IHC, which can affect the results(Ramos-Vara, 2005).
To overcome these obstacles, intravital microscopy techniques are
developed. With these techniques we are able to follow metastasis over time in
vivo, without sacrificing many animals. Furthermore, we can follow one cell or a
group of cells over time and study the behavior of the single cell or group cells in
different environments(Beerling et al., 2011). Taken together, intravital
microscopy is very use full for studying processes, such as metastasis, in vivo to
gain more knowledge to understand such processes better.
Multiphoton microscopy versus other intravital microscopy techniques
There are many intravital imaging techniques, which differ in imaging depth,
resolution, cost and contrast reagents. Ultrasound, MRI and CT are techniques
which are also used in the clinic. Although these techniques have a high imaging
depth, the resolution is very low and only in some parts of the body there will be
a high contrast, but still less details are visible{{3 Reviewed in Beerling,E. 2011}}.
Page | 10
Techniques like positron emission tomography and whole body bioluminescence
are important tools to study metastasis as a process. PET and whole body
bioluminescence both facilitate the discovery of secondary tumors, which are
tissue specific, and primary tumors in several animal models. Furthermore, it is
possible to follow tumor growth in vivo, but these techniques are not able to
follow these processes on the level of single cells(Beerling et al., 2011;
Provenzano, Eliceiri, & Keely, 2009).
Figure 5| Different intravital techniques to visualize metastasis. With these techniques we
can visualize different stages of metastasis. Whole body imaging, PET and
Biofluorescence/Bioluminescence are techniques used in the clinical and cannot follow
metastasis on single cell level. To detect single cells techniques like confocal and multiphoton
microscropy are used(Provenzano et al., 2009).
There are other intravital microscopy techniques, which are able to study
processes, such as metastasis, on the level of a single cell. This can only be done
with the use of contrast reagents(Beerling et al., 2011). These days, there are
animal cancer models, which are able to express fluorescent and
photoswitchable proteins(Beerling et al., 2011; Kedrin et al., 2008). The
expression of these proteins is necessary to visualize tumors or tumor cells, with
the use of confocal laser scanning microscopy and multiphoton
microscopy(Beerling et al., 2011).
Confocal microscopy uses light, ranging from ultra violet to infrared,
which can be focused on a specific part of the specimen. The light of the focused
beam is necessary to excite a photon, which is absorpted by a fluorophore,
resulting in the emission of fluorescence in different directions in the tissue. The
fluorescent signals are collected by the pinhole aperture and the signals which
are from outside the field are blocked(Smith, 2001). The spinning disk confocal
microscopy uses multiple pinholes to block signals from out of the field(Beerling
et al., 2011; Smith, 2001), which results in the enhancement of contrast and an
increased Z-resolution and this make it easier to visualize the subcellular
structues(Beerling et al., 2011). In addition, there is less photobleaching with the
use of spinning disk confocal microscopy(Smith, 2001), so long term imaging of
tumor cells can be performed as well(Beerling et al., 2011). However, there
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confocal microscopy has its disadvantages too. There is more photobleaching
with the use of another confocal microscopy, the laser scanning confocal
microscope(Smith, 2001). More important is that the confocal microscopy uses
light to excite photons to induce fluorescence. These photons scatter in the
tissue, which can contribute to blurry pictures(Dunn & Young, 2006;
Ntziachristos, 2010; Provenzano et al., 2009) and this limits the imaging depth in
specimen to 100µm(Beerling et al., 2011).
By contrast, multiphoton microscopy makes use of near infrared (NIR)
light, ranging from 700nm to 1100nm, to excite photons(Konig, 2000). In this
techniques photons are used with half the energy as one photon used with
confocal microscopy. Two photons has to be excited, before absorption by a
fluorophore can occur, resulting in fluorescence(Beerling et al., 2011). Because
this is a rare event, it can only occur at the focal point and photon scattering
cannot occur. This results in a deeper penetration of the light within the
specimen to 1000µm(Konig, 2000). With the use of multiphoton microscopy
there is less photobleaching and phototoxicity, making this technique also useful
for long term studies following tumor cells. In addition, multiphoton microscopy
can generate second harmonic generation (SHG) signals. These second harmonic
generation signals can provide essential information about the interaction
between the cell and its surrounding environment, such as collagen(Provenzano
et al., 2009). The generation of these signals do not interfere with the activation
of photons, to induce fluorescence(Beerling et al., 2011). Taken together, both
techniques can be very useful in studying metastasis, but due to the imaging
depth and the high detection effenciency multiphoton microscopy is a good
alternative to study metastasis in vivo (Beerling et al., 2011; Provenzano et al.,
2009).
The role of fluorescent proteins in research
Using multiphoton microscopy or other intravital microscopy techniques, makes
it possible to visualize interactions between different molecules and cell types in
vivo en in vitro. For years, studies using intravital microscopy, were limited in
studying processes such as cell-cell interactions, because cells were labeled with
vital dyes and they could only study the vasculature of the tumor. With the
discovery of the green fluorescent proteins (GFP) and the production of the three
other colors, red fluorescent protein (RFP), cyan fluorescent protein (CFP) and
yellow fluorescent protein (YFP) it is possible to study different cell types in vivo,
specifically tumor cells(Condeelis & Segall, 2003; Hoffman, 2009). Farina et al.
was the first to characterize the behavior of carcinoma cells during migration
with the use of GFP and confocal microscopy(Condeelis & Segall, 2003). The use
of GFP to label tumor cells for a longer period, resulted in questions about the
long term effects. It was unknown if GFP affected the behavior of tumor cells
during a time period. Several studies with GFP models have shown that the
tumor is not affected in processes, like growth, when used for a longer
period(Condeelis & Segall, 2003).
A couple of years ago photoswitchable proteins were discovered, which
are fluorescent proteins as well. With photoswitchable proteins, different cell
types can be labeled and they can be combined with the other fluorescent
proteins to study for example the behavior of tumor cells(Beerling et al., 2011).
Page | 12
The advantage of the use of fluorescent proteins is that it makes it possible to
study the cell fate over a longer period of time(Beerling et al., 2011; Ottobrini,
Martelli, Trabattoni, Clerici, & Lucignani, 2011).
Beside labeling with fluorescent proteins, cells can be labeled with
fluorescent trackers(Beerling et al., 2011; Ottobrini et al., 2011). Cells are
isolated and labeled, before they are transferred back into the animal(Beerling et
al., 2011). Labeling cells with fluorescent trackers has the advantage that
fluorescent trackers easily penetrate the membrane and that they bind specific
targets. The drawbacks of this method are that the rate of successful binding of
the label depends on the cell type (the membrane composition, the phagocytic
capacity of the cells etc.), but more important, these trackers can only be used for
cells which cannot dedifferentiate anymore, such as macrophages and dendritic
cells. If proliferating cells are labeled with these trackers, the label in the
daughter cells might be diluted or lost due to cell cycle events or cell
death(Ottobrini et al., 2011).
The only problem using most fluorescent proteins and dyes
simultaneously, results in the overlap of the emission spectra. To discriminate
between these fluorescent proteins and dyes, is using fluorescence lifetime
imaging microscopy (FLIM). With FLIM the lifetime of the fluorescent signal will
be measured. Furthermore, FLIM is very useful in distinguishing between tissue
types, by measuring the autofluorescence. Autofluorescence occurs when cells
are excitated, resulting in the emission of fluorescence by endogenous
components, like melanin and tryptophan. Measuring the lifetime of the
autofluorescence makes it possible to discriminate between tumor and healthy
tissues(Beerling et al., 2011).
Imaging processes over a longer period
Many studies involving intravital microscopy have focused on the visualization of
tumor cells in vivo. A lot of these studies followed tumor cells for a short period
of time, from 4 hours up to 24 hours, thereby lacking information about tumor
cells which are able to invade the underlying stroma. The technique used to
follow cells in such a small time span is surgical dissection, but this technique has
several disadvantages. The consequences of surgical dissection of the imaging
site are dehydration of the dissected tissue, survival and/or thermoregulatory
control of the animal, side effects of the use of anesthesia for a longer time period
and the site to be imaged can be limited(Kedrin et al., 2008).
Following tumor cells for a longer period, to gain more knowledge about
cell behavior resulted in the development of observation chambers and Sandison
(1924) was the first to implant a chamber into animals. In 1993 Lehr et al.
developed the skin fold chamber, which were implanted on the dorsal side of the
animals. These chambers combined with intravital microscopy are used to study
the microvasculature and growth of non-neoplastic and neoplastic tissue for
several days(Lehr, Leunig, Menger, Nolte, & Messmer, 1993).
After the skin fold chamber, orthotopic chambers are developed to study
tumors in their natural environment. Kedrin et al. developed a mammary
imaging window (MIW) to track subpopulations of tumor cells within different
tumor microenvironments with the use of photo-switchable fluorescent proteins.
Cell migration can be followed for more than one day and the imaging site can be
Page | 13
studied up to 21 days post implantation. With the use of the MIW Kedrin et al.
found that in the tumor there are different microenvironments and that these
environments affect intravasation and invasion of the tumor cells in breast
tumors(Kedrin et al., 2008). So, imaging windows are very useful in studies
about metastasis, to acquire more information about tumor cell behavior in vivo
combined with intravital microscopy.
The role of multiphoton microscopy in metastasis: lymphatic and/or
hematogenous?
When tumor cells are detached from the tumor, they can escape the tumor via
lymphatic or blood vessels. However, there are studies which have shown with
intravital imaging that lymphatic vessels are lacking within the tumor itself or
are non functional(Beerling et al., 2011; Pepper et al., 2003). When the tumor
cells enter the underlying stroma, they are able to metastasis via the lymphatic
system. After entering the lymphatic system, the tumor cell first ends up in the
lymph node where it can stay and grow or go to the heart via the efferent vessels
of the lymphatic system(Beerling et al., 2011; Pepper et al., 2003). From the
heart the cells go with the blood flow to other parts of the body, where secondary
tumors can be formed(Beerling et al., 2011). The detection of tumor cells within
the axillary node is used as an marker for metastasis(Patani & Mokbel, 2011), but
it is still unknown if tumor cells start to metastasize via the lymphatic system or
that this only shows the invasiveness of tumor cells within the lymph
nodes(Beerling et al., 2011). Thus, it is still unknown via which route tumor cells
metastasize to other parts of the body.
To figure out if tumor cells metastasize via the hematogenous route,
lymphatic route or both routes, Giampieri et al. used the transforming growth
factor β (TGFβ) to study intravasation of tumor cells in lymph nodes or blood
vessels. TGFβ can change the cell behavior, by the activation of Smads and Twist,
which results in EMT and a more invasive and mesenchymal character of these
cells. With the use of multiphoton microscopy, Giampieri et al. studied the
intravasation process of MTLn3 cells in mice models. When TGFβ is expressed in
these cells, Smad2, which is bound to GFP, translocates to the nucleus and
accumulate. Within the tumor there was a difference in TGFβ signaling, resulting
in quick moving single cells and slow moving cells, which are moving in groups.
The cells which had high TGFβ signaling, moved toward blood vessels to
intravasate them, before moving to other organs. Furthermore they studied the
behavior of MLTn3 cells when there is no TGFβ activity at all. Overexpression of
the TGFβ type 2 receptor, a dominant negative receptor, resulted in cohesively
cell movement and intravasation into the blood vessels could not occur anymore.
More important to notice is that these cells are still able to intravasate the lymph
nodes to cause lymphatic metastasis and to spread throughout the
body(Giampieri, Pinner, & Sahai, 2010). So, TGFβ is an important player for
intravasation of single tumor cells into the blood vessels and this suggest that the
tumor models used, tumor cells metastasis via the blood circulation. However,
this has to be studied in other cancer models before it is proven that tumors start
metastasizing via the blood circulation and not via the lymphatic system
(Beerling et al., 2011; Giampieri et al., 2010).
Page | 14
The role of multiphoton microscopy in metastasis: invasion and intravasation
As mentioned before, until the development of intravital imaging, most studies
were done with the use of techniques which provides static pictures. These
pictures showed where the tumor cells are located in the tissue or organ and this
provided information to determine whether the patient had a good or poor
prognosis(Beerling et al., 2011). However, tumor cells which are escaping the
tumor via blood vessels within the tumor are not marked in the pictures, because
it was overlooked or not studied yet. Furthermore, it is not possible to visualize
the intravasation and motility of these cells within the tumor(Beerling et al.,
2011). During tumor formation there will be angiogenesis, so tumor cells can
escape the tumor and move to other tissues and organs. The escape via blood
vessels in the tumor mass is easier than the intravasation of mature blood
vessels, because these vessels are very leaky due to the poor structure. The blood
vessels within the tumor lack a monolayer of endothelial cells at the outside of
the vessel and there is loss of interconnections between cells as well. In contrast
the blood vessels in the underlying tissue have a dense basement membrane, so
intravasation of these vessels is harder(McDonald & Baluk, 2002).
Intravital microscopy have shown that tumor cells within the tumor end
up in the blood circulation accidently and some delibaretly, but that there are
tumor cells too, which migrate toward blood vessels in the surrounding
microenvironment. Kedrin et al. showed with the integration of a mammary
imaging window, that activated cells labeled with the photoswitchable protein
Dendra2 show migration and invasion in the surrounding tissue stroma of the
tumor. Futhermore, they showed that the microenvironment does affect the
behavior of tumor cells. In microenvironments without blood vessels
surrounding the photoswitched cells there is barely cell migration and invasion
of the surrounding stroma, but the red cells are increased. Nevertheless, tumor
cells in microenvironments near blood vessels, showed a decrease in tumor cells
and a higher migration towards the blood vessels, resulting in intravasation of
the blood vessels(Kedrin et al., 2008).
Figure 6| Following tumor cell movement, within 24 hours, in two different environments:
with and without blood vessels surrounding tumor cells. (a) The pictures show MLTn3
breast tumor cells, nonphotoswitched(green) and photoswitched(red) in an environment
without blood vessels nearby. There is migration of the tumor cells, but this is limited.
Nonetheless, there are more photoswitched cells visible. (b) In this environment there is a blood
Page | 15
vessel next to the photoswitched tumor cells and most of the cells migrate towards the blood
vessel or to other areas outside the field. The cells next to the blood vessel are decreasing,
suggesting that intravasation occurs into the blood vessel and spreading throughout the
body(Kedrin et al., 2008).
Taken together, with the use of intravital microscopy we are able to visualize the
intravasation of active and inactive tumor cells into blood vessels, which are
localized in the tumor mass. This shows that intravasation of tumor cells into
blood vessels not only occurs during the invasion of the underlying tissue
stroma. So, escaping the tumor mass via the blood vessels within the tumor,
makes this a very good escape route, which can be important to form secondary
tumors elsewhere in the body(Beerling et al., 2011).
Discussion
In this review, I have tried to show that multiphoton microscopy is a good
alternative compared to other intravital micropscopy techniques for studying
cancer and especially metastasis. With this technique we are now able to follow
tumor cells for longer periods in vivo with the use of imaging windows, resulting
in gaining more knowledge about important processes like invasion,
intravasation and tumor cell spread via the lymphatic or hematogenous system.
Although a lot of studies focused on the early steps of metastasis, fewer studies
focused on the behavior of tumor cells within organs, which are targets for
metastatic lesions. If we are able to visualize tumor cells within in organs, such as
the lungs, and to follow these over time with intravital microscopy, this will solve
many questions, like why tumor cells specifically go to the lungs to form
metastasic lesions or why tumor cells enter the dormant state and how to escape
this state(Beerling et al., 2011).
Although multiphoton microscopy has overcome most disadvantages of
the other intravital imaging techniques, this technique also has disadvantages.
Some disadvantages are that the costs are very expensive and the imaging depth
is only <1000µm. For the future it is important to try to overcome these
limitations, so studying processes like metastasis is easier, especially if you want
to look at tumors which are located at >1000µm depth(Provenzano et al., 2009).
Unfortunately, the use of intravital imaging techniques cannot provide
information about the molecular pathways, which are involved in these
processes. Studying metastasis or tumor formation is now mainly observational
instead of providing information about molecular pathways and it is important
for the future to use multiphoton microscopy in experiments, which give more
information about the molecular processes. To study the molecular mechanisms
intravital microscopy can be combined with other techniques like FRET and
FLIM. These techniques are used to study processes, like intracellular signaling
and communication between cells and this can contribute a lot to metastasis
research(Beerling et al., 2011; Provenzano et al., 2009). The combination with
other techniques can also help solving questions about other processes, not
involved in metastasis or tumor formation. Although there are some good results
with the use of FRET/FLIM together with multiphoton microscopy, still there are
some disadvantages. It is known that a lot of FRET biosensors are not sensitive
enough for the use in multiphoton microscopy experiments and the
Page | 16
incorporation of these probes. Another problem is the low signal-to-noise ratio
and the ambition is to increase the sensitivity and signal-to-noise ratio for the
new probes, so that the results of the experiments are better. These probes are
now made for studying e.g. cell motility, but the aim is to make new probes for
other processes like adhesion or proliferation(Beerling et al., 2011).
Multiphoton microscopy is a key player in understanding the process of
tumor formation and especially metastasis. This technique can give us more
information about essential steps during metastasis, like invasion, migration and
extravasation. Nowadays, the technique is only used to observe how metastasis
takes place, but does not give any information about the molecular pathways
involved. In addition, most models used for studying cancer make use of
xenograft techniques, so the environment of the tumor differs from the natural
environment. In the tumor environment there are many key players, like
immune cells, which are involved in the process of tumor formation and
metastasis. Fortunately there are some cancer models, which are mimicking the
development of human tumors. Taken together, multiphoton microscopy is a
good alternative compared to other intravital techniques for studying metastasis,
to understand the process better and to provide treatment techniques to prevent
the formation of secondary and/or tertiary tumors(Beerling et al., 2011).
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