AAA Postdoctoral Fellowship
Investigating Eph/Ephrin signaling during Melanoma Metastasis in vivo
ABSTRACT
Currently, the lifetime risk for developing cancer is just over 40% and it is predicted that
nearly 600,000 Americans will die of cancer this year. Importantly, it is estimated that 90% of all
cancer-related deaths can be ascribed to metastasis. Metastasis highlights a complex, dynamic
relationship between the tumor and its microenvironment. Microenvironmental signals that
regulate or inhibit metastatic behaviors have been slow to discover due to challenges
associated with studying the metastatic process in vivo.
Our laboratory has developed an in vivo chick embryo model to study complex molecular
and behavioral traits associated with metastasis. I present preliminary data suggesting that
guidance receptors, namely Eph receptor tyrosine kinases, may be involved in the metastatic
step of intravasation and that their expression patterns may define a metastatic signature. The
affirmation of our hypothesis carries both prognostic and therapeutic implications.
SPECIFIC GOALS AND SIGNIFICANCE
My long-term research goal is to identify signatures of metastatic progression that
predict precise cell behaviors from gene expression profiles. As an NIH Ruth Kirschstein
Postdoctoral Fellow (2010-2013), I developed the chick embryo as a multi-faceted model
system, using in vivo dynamic imaging and novel gene profiling techniques, to visualize
metastatic melanoma behaviors and define molecular signatures of small numbers of
transplanted melanoma cells into the chick embryonic neural crest microenvironment.
The focus of my study is to investigate the roles of Eph receptor tyrosine kinase
receptors (and ligands) in sensing and responding to microenvironmental cues that influence
metastasis. The ratification of my hypothesis carries significant prognostic and therapeutic
implications. First, expression patterns of these neural crest guidance receptors may uniquely
identify cells with metastatic potential, providing novel prognostic tools to clinicians. Second,
the molecular mechanisms associated with guidance receptor function may elucidate specific
processes of the metastatic cascade amenable to therapeutic targeting.
BACKGROUND AND PRELIMINARY DATA
Melanoma represents one of
the most aggressive solid tumors
and occurs following the neoplastic
transformation of melanocytes.
Melanocytes derive from a highly
invasive and multi-potent embryonic
cell population called the neural
crest. As such, we tested the
hypothesis that melanoma (a cancer
of melanocytes) is intrinsically
predisposed to aggressive,
Figure 1. This graphical abstract represents our hypothesis that
metastatic behaviors resulting from
melanoma metastasis is promoted by the aberrant expression of
its ancestral relationship to the
neural-crest-related genes.
neural crest (Figure 1). To test
this, we transplanted small clusters of C8161 human metastatic melanoma cells into the chick
embryonic neural crest microenvironment. We observed that melanoma cells responded to host
embryonic neural crest cues by migrating along canonical neural crest migratory routes and
populating neural crest target tissues (Kulesa et al., 2006). Since that time, we have been
working to understand the powerful effects of the embryonic microenvironment in dictating
melanoma cell migratory behaviors and gene expression patterns.
AAA Postdoctoral Fellowship
As these initial findings supported our hypothesis that
melanoma cells exploit an intrinsic, neural crest-related
migration program to drive metastasis, we next analyzed gene
expression patterns of a number of neural crest-related genes
in C8161 cells following transplantation into the neural crest
microenvironment (citation). We observed that C8161 cells
responded to the neural crest microenvironment by upregulating several classes of cell surface receptors responsible
for sensing and responding to microenvironmental cues.
These gene families included Ephs and Ephrins, Plexins and
Semaphorins, Neuropilins, Slits and Robos. In contrast,
primary melanocytes did not up-regulate any of these genes
(citation). All of these gene families are involved in cell
guidance during embryonic development, but their roles in
adult tissues or cancer pathogenesis have been less
characterized.
My work has identified altered expression patterns for
Figure 2. Re-expression of
Eph/Ephrin family members in C8161 cells compared to
EphB6 in C8161 melanoma cells
results in aberrant directional
primary melanocytes (citation). Among these, I showed that
migration without affecting
EphB6 gene expression was silenced in C8161
migratory ability. EphB6cells but expressed by primary melanocytes. Since loss of
expressing melanoma cells
EphB6 is associated with a higher rate of metastasis and poor
deviated from the typical neural
crest migratory route, suggesting an
clinical outcome in several different cancer types, I
altered cell-microenvironment
investigated whether the loss of EphB6 affects melanoma cell
interaction.
invasion and metastasis. First, I re-expressed EphB6 in
C8161 cells and transplanted these cells into the chick embryo neural crest microenvironment
(hindbrain at rhombomere 4 (r4), 10-somite stage). Whereas parental C8161 cells typically
migrate along the canonical neural crest migratory stream lateral to r4, EphB6+ cells were
observed streaming en masse toward the area adjacent to r5,
an area typically inhibitory to neural crest migration (Figure
2A-D). Interestingly, total migratory length was unaffected
(Figure 2E-F).
Following these observations, we next tested whether
EphB6 re-expression affected metastasis. Using a CAM
metastasis assay that specifically assesses the rate of
intravasation, I observed a significant reduction in metastatic
potential in EphB6+ C8161 cells (Figure 3).
Loss of cell directionality and reduced metastatic
potential argue for a distinct role for EphB6 during
metastasis. I postulate that re-expression of EphB6 forces
the tumor cell to recognize inhibitory signals present within
the microenvironment. My proposed study will test the
following hypothesis: metastatic cells are guided and driven
Figure 3. The CAM metastasis
assay demonstrates that reby dysregulated Eph/Ephrin signaling that allows tumor cells
expression of EphB6 in C8161
to overcome inherent microenvironmental obstacles.
melanoma cells causes a
The success of my fellowship carries both
significant loss of metastatic
prognostic
and therapeutic implications. First, expression
potential. Using this assay, tumors
patterns
of
guidance receptors implicated in the metastatic
can be grown on the vascular CAM
tissue, visualized for cell dynamics,
process may uniquely identify cells with metastatic potential,
and evaluated for metastatic
providing novel diagnostic and prognostic tools to clinicians.
potential using 2-photon microscopy
Second, the molecular mechanisms associated with
and highly sensitive qPCR.
AAA Postdoctoral Fellowship
guidance receptor function may elucidate specific processes of the metastatic cascade
amenable to therapeutic targeting.
In addition to the possible clinical implications, the proposed work will also benefit the
scientific community by developing and modernizing techniques, including the chorioallantoic
membrane (CAM) metastasis assay, to improve the visualization and investigation of complex
tumor cell behaviors in vivo.
RESEARCH DESIGN
Specific Aim 1 – Develop the chorioallantoic membrane (CAM) assay for 2-photon multispectral microscopy to visualize early steps of the metastatic cascade. Current animal
models of metastasis can be time-consuming, expensive, and are not typically amenable to
visualization with single cell resolution. The embryonic chick CAM provides a highly vascular in
vivo substrate similar to a human microenvironment and accessible to observation of the
complex tumor cell behaviors associated with the metastatic cascade. Development of this
assay would overcome the technical roadblock to visualize the metastatic steps of delamination,
invasion through the extracellular matrix, and intravasation (transendothelial migration) into the
host vasculature of single metastatic melanoma cells. Thus, my goal for this aim is to enhance
a 2-photon non-linear optics-based platform to visualize complex tumor cell behaviors on the
CAM (in vivo) with unprecedented resolution. The in vivo dynamic imaging platform is built
around a Zeiss 780 microscope equipped with a Coherent 2-photon laser, an environmental
chamber, and high sensitivity detectors that allow for deep tissue, multi-spectral imaging (Fig.
4). I have been intimately involved in developing and using this platform for my melanoma
metastasis studies, and together with my PI (x), have recently published a protocol and reviews
of live in ovo imaging that demonstrate the innovation and
novelty of this approach [citation]
The successful completion of Aim 1 will enhance our
state-of-the-art dynamic in vivo imaging platform to include
visualization of complex 3D metastatic tumor cell dynamics in the
highly vascularized CAM.
Specific Aim 2 – Determine the specific Eph/Ephrin interaction(s)
induced by EphB6 that regulate the intravasation process.
Following engagement with an Ephrin ligand, Eph receptor activation
typically induces repulsion away from the Ephrin-expressing cell. I
postulate that the re-expression of EphB6 induces a repulsive
interaction between the tumor cell and the chick endothelium
comprising the CAM vasculature, effectively blocking the metastatic
step of intravasation. My goals for this aim are twofold: a) to
determine the specific Eph/Ephrin interaction(s) induced by EphB6
that regulate the intravasation process and b) evaluate whether
Eph/Ephrin expression patterns define a signature of metastasis.
Pursuant to the completion of this aim, I will be able to address the
following questions:
 Which Ephs and Ephrins expressed by the chick endothelial
cells promote the repulsive response?
 Does EphB6, which is kinase-null, drive the anti-metastatic
response in conjunction with or independent of other kinaseactive Eph receptors?
 Can Eph/Ephrin gene expression patterns predict cells with the
Figure 4. The imaging
platform currently used
for dynamic in ovo
imaging. 2-Photon
excitation provides deep
tissue, multi-spectral
capabilities with high
resolution optics.
AAA Postdoctoral Fellowship
ability to intravasate (and therefore predict metastasis)?
To answer these questions, I will use a combination of imaging, molecular, and biochemical
techniques. First, I will employ an RT-qPCR approach to profile the expression patterns of Ephs
and Ephrins on the chick CAM endothelium. I will then compare the Eph/Ephrin expression
profile of chick CAM endothelial cells to that of the C8161 cells (previously published, citation)
and identify putative receptor-ligand interactions that may be involved in repelling the tumor
cells away from the endothelial cells.
Next, I will utilize the imaging platform described under Specific Aim 1 to specifically
visualize the interaction between tumor cells and the chick CAM endothelium. To identify
possible protein interactions and dimerizations between EphB6 and other Ephs and/or Ephrins, I
will employ co-immuno-precipitation, Western blotting, and immunofluorescense microscopy.
Putative protein-protein interactions will be verified by Forster Resonance Energy Transfer
(FRET) microscopy. Western blotting with phospho-specific antibodies will allow me to evaluate
Eph kinase activation and function.
Following the identification of candidate receptor-ligand interactions altered by the reexpression of EphB6, I will use knock-in and knock-down strategies to confirm gene
involvement. Using my imaging platform, I will be able to visualize, and thus confirm, specific
Eph/Ephrin function in regulating the metastatic process of intravasation. This process will
eventually allow me to identify a signature of metastasis that can then be used to interrogate
other melanomas and, in the future, other solid tumors.
These studies will be the first to investigate the role of EphB6 and Eph/Ephrin signaling
in regulating the metastatic process of intravasation. I postulate that EphB6 forms a
heteromeric complex with one or more Eph receptors and either activates or alters its specific
attractive/repulsive response. I expect that the successful completion of Aim 2 will
demonstrate a vital role for EphB6 in regulating intravasation of tumor cells into blood or
lymph vessels. This may be directly attributable to attractive and/or repulsive cues
mediated by cell-cell Eph/Ephrin interactions and may yield novel therapeutic strategies
focused on disrupting the metastatic process
SUMMARY
In summary, I postulate that metastasis is facilitated by dysregulated cell surface
receptors responsible for sensing and responding to the microenvironment. Specifically, loss of
EphB6 allows a permissive interaction between the tumor cell and the endothelium, allowing
intravasation, while the re-expression of EphB6 restores repulsion and blocks intravasation
(Figure 5 – model). I predict that specific Eph/Ephrin expression patterns may define a
metastatic signature for melanoma. It is also possible that these mechanisms are conserved
and that similar signatures may be present in other metastatic tumor types.
Figure 5. A model depicting the development and use of the CAM assay to visualize and investigate complex
tumor cell behaviors in vivo. The model depicts my hypothesis that EphB6 re-expression on metastatic melanoma
cells blocks intravasation by inducing a repulsive Eph/Ephrin signaling response.