DESCRIPTION (provided by applicant): The objective of this application is to develop the career of Dr. Aron Parekh as an independent researcher in the field of cancer invasion. The career development plan created by the PI and his mentor Alissa M. Weaver, M.D., Ph.D., and co-mentor Vito Quaranta, M.D., combines both the didactic and laboratory training required to build Dr. Parekh's biological knowledge and skills. The ultimate goal of the training period is to combine the PI's expertise in mechanics and mechanobiology with the mentors' expertise in molecular biology and advanced imaging techniques to equip Dr. Parekh to become a multidisciplinary researcher focused on the mechanisms of cancer cell mechanobiology. The proposed training opportunity would provide the PI protected time to gain valuable knowledge and experience in acquiring these skills to position him for a successful career in cancer invasion. The research proposal, described below, serves as the foundation of this five year career development plan and of future funding proposals. Long-term clinical outcomes are dependent on whether carcinoma cells leave the primary tumor site by migrating and invading through epithelial and adjacent stromal tissues. Cancer aggressiveness has been linked to tissue density and rigidity both in vivo and in vitro. Our laboratory has shown that mechanosensing of rigid substrates is correlated to increased extracellular matrix (ECM) degradation due to elevated activity of invadopodia, the cytoskeletal structures thought to be critical for proteolytic invasion. Therefore, these results suggest that mechanical factors such as tensile forces play an important role in driving a malignant phenotype. However, the link between biomechanics and the invasion of actual tissues remains inconclusive due to the lack of in vitro models that mimic true tissue properties, particularly those of the epithelial basement membrane (BM) and stroma. In vivo, mechanical forces generated by tumor cell packing and the desmoplastic stroma lead to increased rigidity or tension in the mammary gland and mechanical loading of the local ECM including the BM and adjoining stroma. Therefore, mechanical forces exerted on the local ECM may play an important role in regulating the invasive phenotype, but currently no studies have tested the role of ECM biomechanics under conditions that simulate the tumor microenvironment. The goal is to determine the role of these external forces in regulating cancer cell migration and invasion through the ECM. To achieve this goal, three specific aims are proposed. In Specific Aim 1, the chemical, physical, and mechanical properties of the ECM scaffold urinary bladder matrix-BM (UBM-BM) will be characterized to establish this material as a physiologically relevant in vitro ECM model. In Specific Aim 2, the hypothesis that BM tension activates a malignant phenotype that facilitates invasion will be tested by examining the penetration of UBM-BM under tension by invasive cancer cells. In contrast, Specific Aim 3 will test whether invasion through the adjacent stromal tissue occurs independent of proteolytic degradation and/or mechanosensing by utilizing the connective tissue component of UBM-BM as a model for the stroma. Dr. Parekh anticipates that these studies will yield important insight into the mechanism responsible for mechanically activating cancer cells to penetrate the BM and stroma and could open the door for novel therapeutic strategies that interfere with these processes.

PUBLIC HEALTH RELEVANCE: Project Narrative Cancer cells that originate in the breast and invade other parts of the body continue to be a challenging clinical problem that severely affects the long-term survival rates of patients. Mechanical factors are now thought to play a crucial role in driving cancer cells to leave the primary tumor site, invade neighboring tissue, and eventually spread throughout the body. In order to develop new medical therapies to prevent cancer cell invasion and significantly impact patient outcomes, the mechanism by which mechanical factors regulate cancer cell invasion must first be elucidated. Project Narrative Cancer cells that originate in the breast and invade other parts of the body continue to be a challenging clinical problem that severely affects the long-term survival rates of patients. Mechanical factors are now thought to play a crucial role in driving cancer cells to leave the primary tumor site, invade neighboring tissue, and eventually spread throughout the body. In order to develop new medical therapies to prevent cancer cell invasion and significantly impact patient outcomes, the mechanism by which mechanical factors regulate cancer cell invasion must first be elucidated. __SpecificAimsTextDelimiter__ 10. Specific Aims Metastases resulting from invasive breast cancers originating from mammary gland carcinomas continue to impact long-term survival rates. Cancer aggressiveness is strongly associated with tissue density both clinically and in animal models, and numerous studies utilizing in vitro extracellular matrices (ECMs) have shown that mechanical factors such as rigidity and density can regulate cancer cell invasion. In vitro work from our laboratory has linked mechanosensing of rigid substrates by molecules such as focal adhesion kinase (FAK) to increased ECM degradation at cytoskeletal structures known as invadopodia. Although these results suggest that mechanical forces play a pivotal role in driving malignancy, the link between biomechanics and invasion remains inconclusive due to the lack of in vitro models that mimic the complex physical and mechanical properties of the ECM in vivo, particularly the epithelial basement membrane (BM) [and underlying stroma]. Therefore, further studies are required utilizing physiologically relevant models to properly elucidate this relationship in order to develop novel therapeutic strategies for preventing invasion. Mechanical forces are thought to be a critical component of malignant transformation of the breast. As tumor growth progresses, mammary gland tension increases due to mechanical forces generated by tumor cell packing and stromal deposition and crosslinking. Increased tension leads to mechanical loading of the local ECM and subsequent activation of transformed cells via mechanosensing. [The microenvironment at the tumor-ECM interface consists of the BM and underlying stromal tissue. The BM is a tight protein network that serves as the primary barrier to cancer cell invasion. Once the BM is breached, cells must then navigate the adjacent stroma to reach the vasculature for metastases to occur.] Penetration of the [ECM] is facilitated by protease degradation, particularly by membrane-type-1 matrix metalloproteinase (MT1-MMP) which is found at invadopodia. Therefore, mechanical loading of the [ECM] must play a crucial role in regulating the invasive phenotype. However, no studies have tested the means by which cancer cells transmigrate a true BM [or stroma] under conditions that simulate the tumor microenvironment during tumorigenesis. My goal is to determine the role of external mechanical forces in regulating cancer cell migration and invasion through [these ECM layers. I propose three aims to explore invasive behavior through the BM and stroma to determine the biomechanical invasion mechanisms pertinent to each tissue utilizing a novel in vitro model:] Specific Aim #1: To characterize the chemical, physical, and mechanical properties of urinary bladder matrix-basement membrane (UBM-BM). The mechanical properties of biological tissues depend on their chemical composition and physical characteristics and can change in response to external forces. These properties influence the invasive phenotype but are not appropriately represented by current in vitro models which do not capture all of the unique properties of the BM [and stroma.] Therefore, I will establish UBM-BM, a decellularized ECM scaffold, as a physiologically relevant in vitro model by characterizing these properties using immunohistochemistry, electron microscopy, swelling experiments, and mechanical testing. Specific Aim #2: To test the hypothesis that BM tension activates a malignant phenotype that facilitates invasion. Cells detect mechanical properties and forces through mechanosensing proteins like FAK, and these signals lead to increased degradation by MT1-MMP at invadopodia. Therefore, I will simulate the tension generated in the tumor microenvironment by stretching UBM-BM and identify the resulting cancer cell phenotype with immunofluorescence for activated FAK, MT1-MMP, and invadopodia. BM penetration will be evaluated with electron microscopy, immunohistochemistry, and Western blotting. In addition, I will utilize chemical inhibitors and FAK and MT1-MMP knockdowns to establish the need for mechanosensing and proteolytic degradation in BM transmigration. The objective is to determine whether BM tension increases mechanosensing and proteolytic degradation to enhance BM invasion. [Specific Aim 3#: To determine whether invasion through the adjacent stromal tissue requires proteolytic degradation and mechanosensing. In contrast to the thin, planar, and highly crosslinked BM structure, the stroma is composed of a looser three-dimensional network of collagen fibers. Due to these differences in structural properties, a critical question in the cancer biology field is whether stromal invasion occurs independent of proteolytic degradation and/or mechanosensing especially when considering the tensile forces that are exerted on the local ECM in vivo during tumorigenesis. I will utilize the techniques in Specific Aim 2 to determine the requirement for proteolytic degradation of stromal ECM by migrating cancer cells as well as the role of mechanosensing. The objective is to determine whether tensile forces activate an invasive phenotype that requires proteolytic degradation and/or mechanosensing to navigate the adjacent stromal tissue.]