ABSTRACT Metastasis, the primary cause of breast cancer-related mortality, is a multistep process culminating with the formation of tumor foci within distant organs. However, only a subpopulation of cancer cells within the primary tumor microenvironment is capable of completing the entire metastatic cascade, which includes intravasation, survival in circulation, extravasation, and tumor growth at distant sites. Currently, there are no curative treatments for metastatic breast cancer. Understanding the factors that induce a pro-metastatic cancer cell phenotype and cancer cell dissemination mechanisms is key to developing life-saving therapies against this deadly disease. We identified a population of highly invasive, non-proliferating, non-apoptotic, chemo-resistant cancer cells capable of intravasation. These cells express high levels of MenaINV, a pro-metastatic isoform of the actin-regulatory protein Mena, and low levels of the anti-metastatic isoform, Mena11a. We found that MenaINV expression (published) and a stem cell program (preliminary results) are induced by Notch signaling in tumor cells by direct contact with tumor-associated macrophages. The emergence of MenaINV-High/Mena11aLow stem cells may be one of the crucial steps to metastasis because these cells are not only intravasation- competent but also have tumor-initiating capability. In primary breast tumors, cancer cells expressing MenaINV- High/Mena11aLow are able to enter blood vessels through Tumor Microenvironments of Metastasis (TMEM) doorways. These tightly controlled transient openings in capillary walls were first described by our group and are composed of macrophages, endothelial cells and Mena-expressing tumor cells in direct physical contact. Similar micro-anatomical structures are also observed in lung metastases, but the dissemination mechanism from this secondary site is currently unknown. Interestingly, we found that chemotherapy induces co- expression of MenaINV and stem cell transcription factor Sox9 in tumor cells through a macrophage-dependent mechanism. Importantly, we and others found that chemotherapy also increases the density of TMEM doorways. Thus, we hypothesize that tumor-associated macrophages induce a pro-metastatic cancer cell phenotype in primary tumors and metastatic foci, enabling them to disseminate via TMEM doorways, and that this process is potentiated by chemotherapy. We aim to delineate the involvement of NF-kB and Notch in co- induction of invasive (MenaINV-High) and stem phenotypes in both primary tumor and lung metastases in vivo, evaluate cancer cell dissemination mechanisms in lung metastases and evaluate the effect of chemotherapy on cancer cell re-dissemination from lung metastasis and co-activation of stem and MenaINV-High phenotype. Our findings will provide mechanistic insights into the effects of the tumor microenvironment on the induction of metastasis and cancer cell re-dissemination from metastatic foci. This will enable us to identify molecular targets for future therapies that could be combined with chemotherapy to improve outcomes for patients with metastatic disease which would be a major advance in the battle against breast cancer.

PROJECT NARRATIVE Metastatic disease, the most common cause of breast cancer-related mortality, is mediated by cancer cells capable of disseminating and colonizing distant organs. We will determine the signaling events within tumor microenvironment required for emergence of disseminating cancer cell phenotype (invasive and stem). We will also determine the mechanisms by which these cancer cells re-disseminate from metastatic foci, a process that may perpetuate metastases even after the removal of the primary tumor. Since the majority of patients with metastases are treated with chemotherapy we will determine the effect of chemotherapy on these two processes. To capture the true biology of the tumor microenvironment we will employ novel technologies for in vivo imaging of large volumes of tissue along with our novel biosensors, both in primary tumors and in the lungs, with single cell resolution, and over many days. The findings will likely lead to new approaches for treatment of metastatic disease which is responsible for 90 percent of cancer related mortality.