Cell invasion and cell plasticity are critical to human development but are also striking features of cancer metastasis. By distributing a multipotent cell type from a place of birth to distal locations, the vertebrate embryo builds organs. In comparison, metastatic tumor cells often acquire a de-differentiated phenotype and migrate away from a primary site to inhabit new microenvironments, disrupting normal organ function. Countless observations of both embryonic cell migration and tumor metastasis have demonstrated complex cell signaling and interactive behaviors that have long confounded scientist and clinician alike. James D. Murray realized the important role of mathematics in biology and developed a unique strategy to address complex biological questions such as these. His work offers a practical template for constructing clear, logical, direct and verifiable models that help to explain complex cell behaviors and direct new experiments. His pioneering work at the interface of development and cancer made significant contributions to glioblastoma cancer and embryonic pattern formation using often simple models with tremendous predictive potential. Here, we provide a brief overview of advances in cell invasion and cell plasticity using the embryonic neural crest and its ancestral relationship to aggressive cancers that put into current context the timeless aspects of his work.

Melanoma pathogenesis from normal neural crest-derived melanocytes is often fatal due to aggressive cell invasion throughout the body. The identification of signals that reprogram de-differentiated, metastatic melanoma cells to a less aggressive and stable phenotype would provide a novel strategy to limit disease progression. In this study, we identify and test the function of developmental signals within the chick embryonic neural crest microenvironment to reprogram and sustain the transition of human metastatic melanoma to a neural crest cell-like phenotype. Results reveal that co-culture of the highly aggressive and metastatic human melanoma cell line C8161 upregulate a marker of melanosome formation (Mart-1) in the presence of embryonic day 3.5 chick trunk dorsal root ganglia. We identify nerve growth factor (NGF) as the signal within this tissue driving Mart-1 re-expression and show that NGF receptors trkA and p75 cooperate to induce Mart-1 re-expression. Furthermore, Mart-1 expressing C8161 cells acquire a gene signature of poorly aggressive C81-61 cells. These data suggest that targeting NGF signaling may yield a novel strategy to reprogram metastatic melanoma toward a benign cell type.

Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we test the function of differential screening-selected gene aberrant in neuroblastoma (DAN), a bone morphogenetic protein (BMP) antagonist we detected by analysis of the chick cranial mesoderm. Our analysis shows that, before neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, and then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells in vivo and in vitro, respectively. In vivo loss of function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Collectively, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with the inhibition of BMP signaling.

Metastasis highlights a complex, dynamic relationship between the tumor and its microenvironment. Signals that regulate or inhibit metastatic behaviors have been slow to discover due to challenges associated with studying the metastatic process in vivo. Previous work provided data suggesting the involvement of specific cell surface receptors in the metastatic step of intravasation. In order to visualize the process of intravasation in real-time, we worked toward developing a dynamic imaging platform based on the chorioallantoic membrane (CAM) assay. The embryonic chick CAM provides a highly vascular in vivo substrate similar to a human microenvironment and has been employed in metastasis assays previously. However, inherent challenges associated with high resolution time-lapse imaging using established CAM platforms necessitated adaptations to current protocols. As such, we developed a shell-less CAM technique that could dampen the effects of spontaneous movements of the embryo proper, which greatly affect microscopic focus and field of view. These adaptations allowed us to image the interactions between fluorescent labeled tumor cells and unlabeled CAM tissue over time in vivo. In conclusion, we have adaptated the shell-less CAM assay in order to optimize for time-lapse microscopy using any upright microscope. This platform will now allow for the visualization and interrogation of complex tumor cell-endothelial cell interactions characteristic of metastatic intravasation. This research was kindly funded by a postdoctoral fellowship from the American Association of Anatomists awarded to CMB.

Metastatic dissemination drives the high mortality associated with melanoma. However, difficulties in visualizing in vivo cell dynamics during metastatic invasion have limited our understanding of these cell behaviors. Recent evidence has revealed that melanoma cells exploit portions of their ancestral embryonic neural crest emigration program to facilitate invasion. What remains to be determined is how embryonic microenvironmental signals influence invasive melanoma cell behavior, and whether these signals are relevant to human disease. To address these questions, we interrogated the role of the neural crest microenvironment in dictating the spatiotemporal pattern of melanoma cell invasion in the chick embryo using 2-photon time-lapse microscopy. Results reveal that both permissive and inhibitory neural crest microenvironmental signals regulate the timing and direction of melanoma invasion to coincide with the neural crest migration pattern. These cues include bidirectional signaling mediated through the ephrin family of receptor tyrosine kinases. We demonstrate that EphB6 reexpression forces metastatic melanoma cells to deviate from the canonical migration pattern observed in the chick embryo transplant model. Furthermore, EphB6-expressing melanoma cells display significantly reduced metastatic potential in a chorioallantoic membrane (CAM) metastasis assay. These data on melanoma invasion in the embryonic neural crest and CAM microenvironments identify EphB6 as a metastasis suppressor in melanoma, likely acting at the stage of intravasation. Implications: This article links cellular metastasis to behaviors observed in the ancestrally related embryonic neural crest and demonstrates the powerful influence of the embryonic microenvironment in regulating cell migratory behavior. Mol Cancer Res; 12(9); 1303–13. ©2014 AACR.

Metastatic dissemination drives the highmortality associatedwithmelanoma.However, difficulties in visualizing in vivo cell dynamics during metastatic invasion have limited our understanding of these cell behaviors. Recent evidence has revealed that melanoma cells exploit portions of their ancestral embryonic neural crest emigration program to facilitate invasion. What remains to be determined is how embryonic microenvironmental signals influence invasivemelanoma cell behavior, andwhether these signals are relevant to human disease. To address these questions, we interrogated the role of the neural crest microenvironment in dictating the spatiotemporal pattern of melanoma cell invasion in the chick embryo using 2-photon time-lapse microscopy. Results reveal that both permissive and inhibitory neural crest microenvironmental signals regulate the timing and direction of melanoma invasion to coincide with the neural crest migration pattern. These cues include bidirectional signaling mediated through the ephrin family of receptor tyrosine kinases. We demonstrate that EphB6 reexpression forces metastatic melanoma cells to deviate from the canonical migration pattern observed in the chick embryo transplant model. Furthermore, EphB6-expressing melanoma cells display significantly reduced metastatic potential in a chorioallantoic membrane (CAM) metastasis assay. These data on melanoma invasion in the embryonic neural crest and CAM microenvironments identify EphB6 as a metastasis suppressor in melanoma, likely acting at the stage of

We hypothesize that melanoma metastasis mimics aspects of the emigration program of its ancestral cell type, the embryonic neural crest. To test our hypothesis, we performed a molecular comparison between melanoma and the neural crest. Our study revealed that melanoma cells exploit portions of the embryonic neural crest development program to facilitate invasion. In particular, the purported metastasis suppressor EphB6 was reduced in invasive melanoma cells compared to poorly invasive melanoma cells and primary human melanocytes. Thus, we utilized the chick embryo transplant model system to assay the effects of EphB6 re‐expression in melanoma cells. We report that EphB6 over‐expression alters cell directionality of migrating melanoma cells, while also reducing invasive ability. We confirmed the anti‐metastatic effect of EphB6 in melanoma cells using a chick CAM metastasis assay, while tumor formation appeared unaffected. These results suggest that EphB6 acts specifically as a metastasis suppressor, likely at the metastatic step of intravasation. Our studies also demonstrate the chick embryo as a powerful tool with which to compare and dissect the complex mechanisms of metastasis in vivo that are similar or distinct from normal developmental processes. This work was supported by NRSA 1F32CA144297 (cmb) from the NIH, and by the Stowers Institute for Medical Research. Grant Funding Source : NIH 1R01HD057922

Neural crest (NC) cells undergo an epithelial to mesenchymal transition (EMT) in order to exit from the dorsal neural tube. Similarly, ancestrally related melanoma cells employ an EMT-like event during the initial stages of metastasis to dissociate from surrounding keratinocytes. Whether or not the molecular pathogenesis and cellular dynamics of melanoma metastasis resemble the embryonic NC invasion program is unclear. Here, we highlight advances in our understanding of tumor cell behaviors and plasticity, focusing on the relationship between melanoma and the NC invasion programs. We summarize recent discoveries of NC cell guidance and emerging in vivo imaging strategies that permit single cell resolution of fluorescently labeled tumor cells, with a focus on our recently developed in vivo chick embryo transplant model. Crucial to the molecular pathogenesis of metastasis, we highlight advances in gene profiling of small cell numbers, including our novel ability to gather gene expression information during distinct stages of melanoma invasion. Lastly, we present preliminary details of a comparison of specific genetic pathways associated with the early phases of melanoma invasion and known NC induction and migration signals. Our results suggest that malignant melanoma cells hijack portions of the NC program to promote plasticity and facilitate metastasis. In summary, there is considerable power in combining an in vivo model system with molecular analysis of gene expression, within the context of established developmental signaling pathways, to identify and study the molecular mechanisms of metastasis.

In an attempt to elucidate the mechanisms of melanoma metastasis, our laboratory has recently performed a molecular comparison between melanoma and the embryonic neural crest. The neural crest is a multi-potent and highly invasive cell population, and represents the ancestral cell type of melanocytes. We report that melanoma cells exploit portions of the embryonic neural crest development program to facilitate invasion. In particular, Eph receptor tyrosine kinases and their cognate ligands, the ephrins, are dynamically regulated by C8161 melanoma cells to facilitate migration. We noted that the purported metastasis suppressor EphB6 was significantly reduced in invasive melanoma cells compared to poorly invasive melanoma cells and primary human melanocytes. In the current study, we have utilized the chick embryo transplant model system to examine the effects of EphB6 re-expression in C8161 melanoma cells. EphB6 over-expression significantly alters cell directionality of migrating melanoma cells within the embryo. We confirmed the anti-metastatic effect of EphB6 in melanoma cells using a chick CAM metastasis assay, which demonstrated a significant decrease in metastatic potential. Importantly, tumor formation appeared unaffected. These results suggest that EphB6 acts specifically as a metastasis suppressor, likely at the metastatic step of intravasation. Our studies also demonstrate the chick embryo as a powerful tool with which to compare and dissect the complex mechanisms of metastasis in vivo that are similar or distinct from normal developmental processes. Citation Format: Caleb M. Bailey, Paul M. Kulesa. Unveiling the mechanisms of melanoma metastasis using a chick embryo neural crest model system. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B38.

Cancer cells must regulate plasticity and invasion to survive and metastasize. However, the identification of targetable mechanisms to inhibit metastasis has been slow. Signaling programs that drive stem and progenitor cells during normal development offer an inroad to discover mechanisms common to metastasis. Using a chick embryo transplant model, we have compared molecular signaling programs of melanoma and their embryonic progenitors, the neural crest. We report that malignant melanoma cells hijack portions of the embryonic neural crest invasion program. Genes associated with neural crest induction, delamination, and migration are dynamically regulated by melanoma cells exposed to an embryonic neural crest microenvironment. Specifically, we demonstrate that metastatic melanoma cells exploit neural crest-related receptor tyrosine kinases to increase plasticity and facilitate invasion while primary melanocytes may actively suppress these responses under the same microenvironmental conditions. We conclude that aberrant regulation of neural crest developmental genes promotes plasticity and invasiveness in malignant melanoma.

The dynamic nature of the developing embryo makes it challenging to understand complex morphogenetic events using information from large-scale gene expression patterns. What would be more insightful is molecular profiling of small numbers of cells selectively surveyed at specific developmental stages. However, detecting gene expression profile information from small numbers of cells (<10) in homogenous tissue has remained a major challenge. Here, we describe the use of laser capture microdissection (LCM), immunohistochemistry (IHC), and RT-qPCR to extract gene profile information in distinct embryo tissue more precisely than is possible with any other method. We use the chick embryo model system and combine electroporation and dual-label IHC to specifically identify cells for harvest by LCM without significant degradation of total RNA. We describe the development of a pre-amplification protocol for small subpopulations of cells to produce sensitive RT-qPCR results. The gene-specific pre-amplification efficiently and linearly amplifies only gene transcripts of interest from the harvested material without the need for RNA isolation. By combining the above techniques with microfluidic RT-qPCR, we robustly analyze the expression of ∼300 genes from as few as 10 cells harvested by LCM. Together, this protocol presents a confident isolation and means of sensitive expression analysis of small cell numbers from tissues and overcomes a technical hurdle that limits gene profiling.

Abstract Melanocytes derive from a highly invasive, multipotent embryonic cell population known as the neural crest. It has been proposed that this ancestral relationship predisposes melanocytes to having metastatic characteristics following neoplastic transformation to melanoma. In support of this hypothesis, we have previously shown that aggressive melanoma cells transplanted into the chick embryonic neural crest microenvironment respond to neural crest signaling mechanisms by emigrating along defined host neural crest pathways. To evaluate whether melanoma cells usurp aspects of the neural crest program typically silenced in differentiated melanocytes, we have employed the chick embryo transplant model coupled with laser capture microdissection to perform an in vivo molecular analysis of more than 70 neural-crest-related genes. Transplanted cells included c8161 aggressive melanoma cells, a poorly aggressive isogenic counterpart termed c81-61, and primary human melanocytes. We demonstrate that all three cell types express many neural-crest-related genes, but that aggressive cells aberrantly express some genes typically lost during melanocyte differentiation. Furthermore, the analysis revealed a significant increase in the ability of aggressive melanoma cells to respond to changing microenvironments by altering gene expression signatures, while transplanted melanocytes appeared to actively suppress these pathways. This increased responsiveness may be attributed to the dynamic regulation of Eph receptor tyrosine kinases and their cognate ephrin ligands, many of which were differentially expressed in aggressive melanoma cells versus poorly aggressive melanoma or primary melanocytes. Thus, metastatic melanoma cells usurp portions of the neural crest program to promote increased invasiveness and plasticity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3315. doi:1538-7445.AM2012-3315

Cell proliferation is crucial to tissue growth and form during embryogenesis, yet dynamic tracking of cell cycle progression and cell position presents a challenging roadblock. We have developed a fluorescent cell cycle indicator and single cell analysis method, called CycleTrak, which allows for better spatiotemporal resolution and quantification of cell cycle phase and cell position than current methods. Our method was developed on the basis of the existing Fucci method. CycleTrak uses a single lentiviral vector that integrates mKO2-hCdt1 (30/120), and a nuclear-localized eGFP reporter. The single vector and nuclear localized fluorescence signals simplify delivery into cells and allow for rapid, automated cell tracking and cell cycle phase readout in single and subpopulations of cells. We validated CycleTrak performance in metastatic melanoma cells and identified novel cell cycle dynamics in vitro and in vivo after transplantation and 3D confocal time-lapse imaging in a living chick embryo.

The plastic phenotype of aggressive melanoma has presented a significant challenge in the detection and targeting of tumor cells exhibiting stem cell-like characteristics. As the molecular signaling pathways underlying tumor cell plasticity become more transparent, our understanding of how to suppress this elusive phenotype will be enhanced. Indeed, we are making progress in identifying critical embryonic pathways, such as the Nodal signaling pathway, that reemerge in aggressive tumor cells - in the absence of regulatory check points. Because Nodal is not expressed by the majority of normal adult tissues, and is over-expressed by aggressive tumor cells, it represents a valuable new therapeutic target. Collectively, we have learned a great deal from studies that focus our attention on the convergence of embryonic and tumorigenic signaling pathways. At this interaction of normal development and tumor formation reside the clues to suppressing cancer progression.

The incidence of melanoma has steadily and sharply risen over the past century, highlighting our lack of understanding of this deadly cancer. Melanocytes, which give rise to melanoma upon neoplastic transformation, derive from a highly invasive, pluripotent embryonic cell population termed the neural crest. Herein we compare and contrast melanoma with its ancestrally‐related neural crest to evaluate the metastatic predisposition of melanoma cells resulting from similarities with the neural crest invasion program. Using a novel chick embryo model, we combine high resolution in vivo imaging with advanced genetic profiling to characterize behavioral and molecular signatures associated with invasive melanoma cells. We demonstrate that aggressive melanoma cells respond to specific host neural crest inductive cues to advance their migration along neural crest pathways. This model system provides a robust in vivo model to study melanoma cell migration and may open doors to the identification of novel molecular markers and targetable cellular pathways involved in the metastatic process. This work was supported by CA121205 and NRSA 1F32CA144297‐01A1 from the NIH/NCI, and the Stowers Institute for Medical Research.

Vertebrate development is best studied in an intact embryo model, but a robust interface between time-lapse microscopy and in vivo embryo health and maintenance can be difficult to achieve in model systems that rely on external factors for life support. This protocol presents a system for in ovo culture and time-lapse imaging of fluorescently labeled cells within living avian embryos, using a Teflon membrane that is oxygen-permeable and liquid-impermeable. The protocol describes the Teflon membrane assembly (the assembly size can be changed to fit smaller eggs, such as those of the quail), its interface with the egg window, and the use of an upright microscope and heated chamber. The use of the system is demonstrated in chick embryos by following individual fluorescently labeled neural crest cells, a multipotent stem cell-like population that differentiates into a wide range of derivatives and travels extensively throughout the embryo. By combining in ovo culture with confocal or two-photon four-dimensional time-lapse imaging, embryo health can be maintained for up to 5 d, and neural crest cell behaviors can be visualized for long periods of time (approximately 36 h). This technique has been adapted to study somitogenesis.

Cathepsin D (CatD) is a lysosomal aspartyl endopeptidase originally considered a "house keeping enzyme" involved in the clearance of unwanted proteins. However, recent studies have revealed CatD's involvement in apoptosis and autophagy, thus signifying an important function in the proper development and maintenance of multi-cellular organs. In the mammary gland, matrix degradation and the remodeling process are orchestrated by proteolytic enzymes, but the role of CatD at distinct developmental stages has remained mostly unexplored. Based on our previous studies we sought to address the role of this endopeptidase in mammary gland development and remodeling. By employing a mouse model, we report a previously unidentified participation of CatD in different stages of mammary gland development. Our findings reveal that CatD undergoes distinct protein processing at different stages of mammary gland development, and this customized processing results in differential enzymatic activity (constitutive and low pH activatable) best fitting particular stage(s) of development. In addition, at the onset of involution the N-glycan structure of this endopeptidase switches from a mixed high mannose and hybrid structure to an almost exclusively high mannose type, but reverts back to the original N-glycan composition by day 4 of involution. Our findings illuminate (at least in part) the "raison d'être" for CatD's numerous and highly regulated proteolytic processing steps from the pro-form to the mature enzyme. In the mammary gland, specific cleavage product(s) perform specialized function(s) befitting each stage of remodeling. It is noteworthy that deregulated synthesis, secretion and glycosylation of CatD are hallmarks of cancer progression. Thus, identifying the role of CatD in a dynamic normal tissue undergoing highly regulated cycles of remodeling could provide valuable information illuminating the deregulation of CatD associated with cancer development and metastasis.