Funding Integrated Imaging Program Gruss-Lipper BPC Peter T. Rowley Breast Cancer Scientific Research Projects (Round 5) - Racial Disparity in Pro-Metastatic Breast Cancer Microenvironment and Response to Chemotherapy – Oktay NYS-DOH Abstract Breast cancer in black patients behaves more aggressively and more often comes back at distant sites such as lungs, bone or brain, than in non-black patients. The return of breast cancer at distant sites, called metastasis, causes 90% of deaths from cancer. Our data indicate that the difference in breast cancer outcome is partly attributed to racial differences in the way non-cancerous part of tumor, called “tumor microenvironment” communicates with cancer cells. We found that non-cancerous cells may team up with cancer cells to make “doorways”, called TMEM, through which cancer cells enter bloodstream, disseminate to distant sites, and ultimately kill patients. Cancer cells which use TMEM to disseminate make proteins called MenaCalc. Both TMEM and MenaCalc can be detected in breast cancer samples using standard laboratory protocols. Moreover, TMEM and MenaCalc scores correlate with the development of distant metastasis in patients and can be used to determine which patients will develop metastasis. We have early evidence that breast cancer patients of black compared to non-black race have higher TMEM score and more cancer cells which can disseminate through TMEM. Moreover, chemotherapy may in some instances increase the number of TMEM and disseminating cancer cells. The increase in TMEM caused by chemotherapy can be blocked with an agent that inhibits TMEM activity, currently being tested in patients. Since breast cancer deaths occur primarily due to metastatic disease the difference in TMEM-mediated cancer cell dissemination between black and non-black patients may contribute to the disparity in breast cancer outcome. Thus, we will analyze how the number of TMEM doorways and metastasizing cancer cells correlate with distant metastasis and how chemotherapy affects tumor microenvironment in patients of different race. This will help us identify steps in cancer cell dissemination that are more prevalent in blacks, for which specific therapies can be made. Peter T. Rowley Breast Cancer Scientific Research Projects (Round 5) -Direct Visualization of Physiologically Relevant Metastasis Using Surgical and Optical Engineering - Entenberg NYS-DOH Abstract We have developed novel technologies that enable, for the first time, the direct visualization of large volumes of tissue in the living, breathing lungs, with single cell resolution over days to weeks. We will use these technologies to examine tumor cells, in vivo, as they disseminate from primary tumors to the lung vasculature. By using tumor cells with genetically encoded fluorescent biosensors for the expression of stemness associated proteins, we can determine how stemness (tumor initiating capacity) influences the efficiency with which tumor cells progress through each step of the metastatic cascade. Further, our preliminary evidence indicates that macrophages play a role in supporting the survival and retention of tumor cells that reach the lung vasculature. We will investigate this role by systemically altering overall macrophage numbers or their function to see how the efficiency with which stem and non-stem cells progress through the metastatic cascade is impacted. Metastasis is estimated to be responsible for ~90% of all cancer associated mortality. However, the mechanisms underlying which disseminated tumor cells eventually develop into macro-metastases and what conditions in the primary and secondary sites (i.e. lung) enhance or limit metastatic progression, are still incompletely understood. Successful completion of this project will determine the role of cancer initiating stem cells in the formation of lung metastases and how macrophages can alter this role. This has the potential to identify new therapeutics that specifically target the process of metastasis, eliminating the major cause of death from breast cancer. Breast Cancer Research Foundation - TMEM, MENACalc, and MenaINV as prognostic markers for breast cancer metastasis in a multiethnic population -Rohan The goal of this project is to examine the association of markers of dissemination-competent tumor cells (MenaCalc/MenaINV) and intravasation (TMEM) with risk of distant metastasis in a case-control study nested in a large cohort of breast cancer patients at Montefiore Medical Center. Specifically, we propose to: examine the association of TMEM, MenaCalc and MenaINV with risk of distant metastasis. evaluate TMEM jointly with MenaCalc and/or MenaINV to determine whether these markers combined (TMEM/Mena score) are more strongly associated with risk of distant metastasis than any of the markers alone. TMEM, MenaCalc, and MenaINV as Prognostic and Predictive Markers for Breast Cancer Metastasis – NCI, R01, Rohan & Condeelis) Abstract Breast cancer mortality is largely attributable to systemic, hematogenously-disseminated metastatic disease. Given the limitations of current prognostic criteria, new methods to identify tumors likely to metastasize and respond to therapy are needed. Multiphoton-based intravital imaging has shown that invasive carcinoma cells in rodent mammary tumors intravasate via peri-vascular structures containing a Mena over-expressing tumor cell, a macrophage, and an endothelial cell, all contacting each other. This tri-partite arrangement of cells facilitates entry of carcinoma cells into the blood vessel. We have identified this microenvironment in human breast cancer samples using a triple immunostain for formalin-fixed paraffin-embedded tissue that simultaneously labels the 3 cell types. We call the direct apposition of these 3 cell types “TMEM”, for Tumor MicroEnvironment of Metastasis. Recently, in a cohort of patients at Kaiser Permanente (KP), we showed that TMEM was positively associated with risk of distant metastasis in ER+/HER2- breast cancer independently of IHC4, a composite immunohistochemical score (based on ER, PR, HER2, Ki67) that provides prognostic information comparable to the Oncotype Dx® Recurrence Score (RS). Extension of this work to encompass comparison to other recently developed prognostic markers (e.g., PAM50) is now warranted. For TMEM, the invasive carcinoma cells are identified using a protein marker for invasive and migratory cancer cells called “Mena”, which has multiple splice variants: Mena11a is an anti-metastatic isoform expressed in epithelial-like but not mesenchymal-like tumor cells, while Mena invasive (MenaINV) confers a potent pro-metastatic phenotype in mesenchymal-like tumor cells. Recently, we used multiplex quantitative immunofluorescence to estimate the abundance of Mena lacking its anti-metastatic Mena11a isoform. We showed that this marker, MenaCalc, which reflects the relative amount of epithelial-mesenchymal transition (EMT) that a tumor has undergone so that tumor cells can participate in TMEM assembly and interact with TMEMs, was positively associated with risk of breast cancer death. We now propose to: examine the association of these markers (MenaCalc/MenaINV/TMEM) with risk of distant metastasis both in a case-control study of 600 case-control pairs nested in an expanded KP cohort of 8769 breast cancer cases, and in 1000 breast cancer cases sampled from the B28 trial (which assessed the value of paclitaxel as adjuvant chemotherapy); examine the association with risk of distant metastasis of the strongest TMEM-related marker or marker combination in comparison to and in addition to IHC4 and PAM50 (KP study) and Oncotype Dx® RS and PAM50 (B28 study) [~2/3 of the B28 study sample will have Oncotype Dx® RS results available]); examine whether TMEM-related marker or marker combination predicts response to therapy (in B28); and externally validate in the B28 study population the TMEM/Mena score (the combination of TMEM, MenaCalc, MenaINV most strongly associated with risk) developed in the KP study population. Translational Research Program (TRP) of the Department of Pathology and Laboratory Medicine at NewYork-Presbyterian/Weill Cornell Medical Center - 'Tumor Microenvironment of Metastasis (TMEM) Expression in Invasive Breast Carcinoma and Correlation with Distant Metastasis' – Ginter. Abstract Understanding the identity, location and function of cells in the tumor microenvironment is essential to understanding how they dominate tumor phenotype and contribute to dissemination and metastasis. An example of this is the tri-partite structure TMEM (for Tumor MicroEnvironment of Metastasis) consisting of the juxtaposition of a macrophage, an endothelial cell and a tumor cell which function together to act as the doorway for metastatic dissemination. We have created novel imaging technologies consisting of 1)implantable windows with embedded microfluidics that allow serial high-resolution, single-cell microscopy of the primary and metastatic sites over days to weeks, and 2)multi-modal image alignment technologies to register fixed, stained, tissue sections to each other or to the acquired intravital imaging movies. Light activated valves embedded in the microfluidic system will be used to deliver microenvironment-altering chemical (e.g. hypoxia mimetics, chemotherapy) and biological (e.g. function blocking antibodies) agents and label unmarked tissues with fluorescent antibodies, all while imaging the tissue response in real time. Intravital imaging will be used to image the tissue and provide single-cell resolution images that cover the entire tissue over time spans ranging from seconds, to days, and even weekws. Finally, motile cells will be captured, either by chemo-attraction to microfluidic chambers, or laser capture microdissection after fixation, for further expression profiling. Application of these technologies to the study of primary and secondary lesions an unprecedented ability to probe the identity, location, quantity and function of the cells composing the heterogeneous microenvironment. Further, testing, in vivo, the targeting agents affecting the distribution, function and dynamics of the cells forming TMEM, as well as and the cells with which they interact, will enable the rapid determination of those agents with best therapeutic potential. This work will determine how 1) hypoxia, ECM density, immune cell density and chemotherapy initiate and define tumor heterogeneity in the primary and secondary sites; 2) chemotherapy and known blockers of TMEM assembly and function affect the dynamics of intravasation and dissemination in the primary and secondary sites; and 3) the relationship between cellular behavior and phenotype with cellular identity and location. The techniques utilized in this project are generally applicable and will allow molecular identification, localization and quantification of tumor heterogeneity in many cancers and disease models.
