Imaging RhoC-induced Breast Cancer Invasion and Angiogenesis

Institution: Scripps Research Institute
Investigator(s): Konstantin Stoletov, Ph.D. - Konstantin Stoletov, Ph.D. -
Award Cycle: 2005 (Cycle 11) Grant #: 11FB-0088 Award: $15,725
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

This is a collaboration with: 11FB-0088A -

Initial Award Abstract (2005)
Metastasis is the major cause of poor prognosis in breast cancer patients. Current evidence indicates that tumor cell metastasis requires the activation of cell migration and angiogenic programs, increased vascular permeability, proteolysis of the extracellular matrix (ECM), and decreased cell apoptosis. These key factors allow cancer cells to invade into the surrounding ECM, gain access to the vascular system, and survive and proliferate in distant tissues. We are interested in how the small GTPase, RhoC, affects the cellular processes that contribute to breast cancer metastasis. Rho-family GTPases are molecular switches that couple changes in the extracellular environment to intracellular signal transduction pathways. Recently, RhoC was shown to be upregulated and associated with poor patient outcome in inflammatory breast cancer (IBC). IBC is a highly aggressive and angiogenic form of the disease that affects about 5% of women diagnosed with breast cancer each year. Work with cultured cells demonstrates that overexpression of RhoC increases invasion and proangiogenic factors. Based on these initial findings, we will test the hypothesis that RhoC contributes to IBC by increasing the tumor cells’ ability to migrate in complex tissues and to actively change the structure of tumor-induced blood vessels.

However, understanding how tumor cell invasion and angiogenesis occur in developing tumors has been difficult because existing cancer models do not allow direct visualization in high resolution of these processes within living organisms. Therefore, we have developed a model of cancer that utilizes human tumor cells growing in optically clear Zebrafish. This model will allow tumor growth, angiogenesis, and invasion in response to RhoC expression to be directly visualized and imaged. Using in vivo confocal microscopy, we have monitored in time-lapse tumor formation and remodeling of the vasculature in vivo at the subcellular level. Our initial findings reveal that transplanted human breast tumor cells expressing red fluorescent protein rapidly home to existing blood vessels where they attach and form solid tumors. In these developing tumors, we observed that cancer cells induce remodeling of the existing vessels in a dynamic process that involves permeation of the vessel wall and the formation of loop-like structures that subdivide into channels, forming new blood vessel structures. These distinct structures formed only in close proximity to tumor cells and were dynamic, undergoing continual remodeling during tumor progression. Our findings represent the first high resolution observations of breast tumor formation and tumor-induced vascular remodeling in a living organism.

In this CBCRP-funded fellowship, we will utilize our Zebrafish model to determine how the small GTPase, RhoC, induces breast cancer invasion and angiogenesis. Results from these studies will provide critical information on how amplification of RhoC contributes to breast cancer progression in vivo, which will provide a better understanding of how to design pharmacological and genetic agents to eradicate this disease.


Final Report (2008)
Metastasis is the major cause of death in breast cancer patients, and there are no therapeutic agents available to prevent this process. Currently, metastasis is viewed as a highly dynamic process that occurs in several distinct steps. The most critical metastatic steps - intravasation (entry into the circulation) and extravasation (exit from the circulation into metastatic sites) involve close interaction of invading tumor cells with the host vasculature. Understanding of these tumor cell-blood vessel interactions, has been limited by the inability to visualize invading cancer cell behavior in close vicinity to the blood vessels in vivo.

Here we employed Zebrafish, which are transparent, and high- resolution confocal microscopy to study how human cancer cells overexpressing the metastatic gene, RhoC (a small GTPase signaling protein that promotes reorganization of the actin cytoskeleton and regulate cell shape, attachment, and motility), invade living tissues and intravasate into the vasculature. RhoC is involved in highly metastatic inflammatory breast cancer (IBC), however molecular and cellular mechanisms that underlie IBC's high metastastatic potential are unknown. Using a semi-transparent, transgenic Tg(flil:EGFP) Zebrafish line we developed a novel human tumor xenograft (transplant) model that allowed us to monitor with high resolution human tumor cell behavior at the vascular interface in vivo. We found that RhoC expression in the human breast adenocarcinoma switches the tumor cell migration to primitive amoeboid-like and induces formation of multiple membrane dynamic protrusions and blebs. These features allowed tumor cells to penetrate the vascular wall more efficiently. Importantly, tumor cell penetration occurred exclusively at sites of vascular remodeling and not at regions of stable, preexisting blood vasculature. Tumor cell entry into the vascular lumen required continuous secretion of VEGF (vascular endothelial growth factor) that induces vascular openings, which serve as “portholes” allowing intravasation of RhoC expressing cells. Therefore, we proposed a model in which the early steps of metastasis require two independent processes: (1) dynamic regulation of the actin/myosin cytoskeleton within the tumor cell to form protrusive structures, and (2) vascular permeablization as well as vessel remodeling.

Our findings suggest that either of these processes can serve as an efficient target for development of anti-RhoC mediated metastasis therapeutic agents. We plan to continue our investigation of interaction of invading tumor cells with the host vasculature during metastasis. Specifically, we have expanded our model to it allows us imaging late metastatic steps such as tumor cell extravasation and invasion away from the vasculature. Currently, we are conducting live imaging of the extravasating tumor cells and studying the role of prometastatic genes RhoC, twist and VEGF in this phase of metastasis.


Symposium Abstract (2005)
Metastasis is the major cause of poor prognosis in breast cancer patients. Current evidence indicates that tumor cell metastasis requires the activation of cell migration and angiogenic programs, increased vascular permeability, proteolysis of the extracellular matrix (ECM), and decreased cell apoptosis. However, understanding how tumor cell invasion and angiogenesis occur in developing tumors has been difficult because existing cancer models do not allow direct visualization in high resolution of these processes within living organisms. Therefore, we have developed a model of cancer that utilizes human tumor cells growing in optically clear Zebrafish. This model will allow tumor growth, angiogenesis, and invasion in response to specific oncogene activation to be directly visualized and imaged. Using in vivo confocal microscopy, we have monitored in time-lapse tumor formation and remodeling of the vasculature in vivo at the subcellular level.

Our initial findings reveal that transplanted human breast tumor cells expressing red fluorescent protein rapidly home to existing blood vessels where they attach and form solid tumors. In these developing tumors, we observed that cancer cells induce remodeling of the existing vessels in a dynamic process that involves permeation of the vessel wall and the formation of loop-like structures that subdivide into channels, forming new blood vessel structures. These distinct structures formed only in close proximity to tumor cells and were dynamic, undergoing continual remodeling during tumor progression. Significantly, this model allowed us to visualize and quantify the effect of anti-angiogenic compounds, such as VEGFR inhibitor SU541,6 on tumor growth and angiogenesis by adding these compounds to the fish water. Our findings represent the first high resolution observations of breast tumor formation and tumor-induced vascular remodeling in a living organism. We are currently interested in how known oncogenes, such as Src, PI3K and small GTPase RhoC, affect the cellular processes that contribute to breast cancer metastasis. By overexpressing these proteins in fluorescently labeled breast cancer cells we are able to visualize and quantify their effect on tumor cell invasion and angiogenesis in our Zebrafish model. Our initial findings indicate that Src or RhoC overexpression promotes tumor cells to switch from the less efficient mesenchymal mode of migration to the more efficient amoeboid mode.

Results from these studies will provide critical information on how activation of specific oncogenes contributes to breast cancer progression in vivo, which will provide a better understanding of how to design pharmacological and genetic agents to eradicate this disease.


Symposium Abstract (2007)
Understanding the in vivo mechanisms of tumor cell invasion and metastasis has been severely limited by the inability to image this dynamic process in live animals in high resolution. Progress has also been limited by the inability to visualize the early stages of tumor formation and vascular remodeling. Consequently, little is known about how human cancer cells invade through complex tissues and interact with the newly remodeling vasculature to initiate tumor formation and metastasis. To address these limitations, we have developed a new xenograft model that combines the optical clarity and powerful genetics of zebrafish with high resolution confocal microscopy and GFP technology (fluorescence cell imaging). Using this unique system, we studied the role of human metastatic gene RhoC in promoting breast cancer metastasis. RhoC is overexpressed in highly aggressive inflammatory breast cancer however the exact mechanisms by which it enhances tumor cell metastasis are unknown. We made the important discovery that RhoC facilitates a primitive amoeboid-like migration pattern leading to increased breast cancer cell intravasation. High resolution imaging at the vascular-tumor cell interface revealed that this process is mediated by the protrusion of large membranes processes that penetrate deep into the vessel lumen. This process requires the secretion of the vascular permeability factor VEGF, which disrupts the vessel wall allowing membrane penetration and cell intravasation. Our results provide novel insight into mechanisms of cancer cell invasion and intravasation and provide important new information on how RhoC and VEGF cooperate to facilitate cell metastasis in living tissues. Development of this model also provides a simple and cost effective vertebrate system to investigate the action of anti-cancer and anti-angiogenic agents on cancer progression.