Project Grant F30CA318159

IMPLICATIONS OF COAGULATION AND FIBRINOLYTIC CASCADE ACTIVATION IN OSTEOSARCOMA ANGIOGENESIS AND METASTASIS - PROJECT SUMMARY/ABSTRACT: OSTEOSARCOMA IS THE MOST COMMON PRIMARY MALIGNANT BONE TUMOR IN CHILDREN AND YOUNG ADULTS, YET OUTCOMES FOR PATIENTS WITH THE DISEASE HAVE NOT IMPROVED IN OVER 50 YEARS. THESE PATIENTS OFTEN EXHIBIT A HYPERCOAGULABLE STATE, WITH ELEVATED LEVELS OF FIBRINOGEN (A CIRCULATING COAGULATION PROTEIN) IN THEIR BLOOD. OSTEOSARCOMA CELLS ARE KNOWN TO DIRECTLY ACTIVATE THE PROCOAGULANT ENZYME THROMBIN, WHICH CONVERTS CIRCULATING FIBRINOGEN INTO AN INSOLUBLE FIBRIN MATRIX. UNSURPRISINGLY, CANCER CELLS CAN BE FOUND EMBEDDED WITHIN VENOUS THROMBI RETRIEVED FROM PATIENTS WITH OSTEOSARCOMA. THEREFORE, THROMBIN, FIBRIN(OGEN), AND THE HYPERCOAGULABLE STATE INDUCED BY MALIGNANCY HAVE TRADITIONALLY BEEN VIEWED AS TUMOR PROMOTING IN THE CONTEXT OF OSTEOSARCOMA. IN THE PRELIMINARY WORK PRESENTED IN THIS PROPOSAL, IT WAS INITIALLY HYPOTHESIZED THAT DEPLETING CIRCULATING FIBRINOGEN WOULD INHIBIT OSTEOSARCOMA PROGRESSION AND METASTASIS. HOWEVER, IN DIRECT CONTRAST TO THIS INITIAL HYPOTHESIS, IT WAS FOUND THAT PHARMACOLOGIC DEPLETION OF FIBRINOGEN INCREASED OSTEOSARCOMA TUMOR GROWTH, ANGIOGENESIS, AND OSTEOLYSIS. THESE FINDINGS PRESENT AN APPARENT PARADOX: THROMBIN ACTIVITY IS KNOWN TO PROMOTE OSTEOSARCOMA PROGRESSION, YET FIBRIN(OGEN), THE CANONICAL DOWNSTREAM TARGET OF THROMBIN, APPEARS TO INHIBIT IT. THIS SUGGESTS THAT THROMBIN DRIVES TUMOR PROGRESSION THROUGH NON-CANONICAL SIGNALING MECHANISMS, WHILE FIBRIN REPRESENTS A TUMOR-SUPPRESSIVE BYPRODUCT. THIS WOULD IMPLY THAT OSTEOSARCOMA PROGRESSION NORMALLY REQUIRES ACTIVATION OF THE FIBRINOLYTIC CASCADE TO DEGRADE FIBRIN BARRIERS AND ALLOW FOR INVASION AND NEOANGIOGENESIS. THEREFORE, THE OVERARCHING HYPOTHESIS OF THIS PROPOSAL IS THAT SIMULTANEOUS ACTIVATION OF BOTH THE COAGULATION AND FIBRINOLYTIC CASCADES IN THE TUMOR MICROENVIRONMENT (TME) PROMOTES OSTEOSARCOMA PROGRESSION AND NEOANGIOGENESIS. TO DIRECTLY TEST THIS HYPOTHESIS, AIM 1 WILL DELINEATE THE INDIVIDUAL CONTRIBUTIONS OF FIBRINOGEN, FIBRIN, AND FIBRINOLYSIS IN OSTEOSARCOMA PROGRESSION USING GENETICALLY ENGINEERED MICE THAT LACK FIBRINOGEN, EXPRESS NON- CLEAVABLE FIBRINOGEN, OR LACK THE ENZYMES NEEDED FOR FIBRINOLYSIS. THESE COMPLEMENTARY MODELS WILL DETERMINE WHETHER TUMOR SUPPRESSION ARISES FROM CIRCULATING FIBRINOGEN OR DEPOSITED FIBRIN, AND TEST WHETHER FIBRINOLYSIS IS REQUIRED TO OVERCOME FIBRIN(OGEN)-MEDIATED TUMOR CONTAINMENT. AIM 2 WILL DEFINE HOW COAGULATION AND FIBRINOLYTIC PATHWAYS ARE SPATIALLY ORGANIZED AND MECHANISTICALLY ACTIVATED WITHIN THE OSTEOSARCOMA TME. THIS PROPOSAL LEVERAGES SPATIAL TRANSCRIPTOMICS OF MATCHED PRIMARY AND METASTATIC HUMAN OSTEOSARCOMA SPECIMENS TO MAP CELL-TYPE SPECIFIC EXPRESSION OF COAGULATION AND FIBRINOLYSIS RELATED GENES. COMPLEMENTARY IN VITRO STUDIES IN HUMAN AND MURINE OSTEOSARCOMA CELL LINES WILL DETERMINE HOW THESE PATHWAYS REGULATE PROLIFERATION AND INVASION. TOGETHER, THE PROPOSED WORK WILL GENERATE FOUNDATIONAL INSIGHT INTO HOW HEMOSTATIC DYSREGULATION DRIVES OSTEOSARCOMA PROGRESSION AND METASTASIS, WHILE PREPARING THE APPLICANT FOR A CAREER AS AN INDEPENDENTLY FUNDED ORTHOPEDIC ONCOLOGY SURGEON-SCIENTIST DEDICATED TO IMPROVING THE OUTCOMES OF PATIENTS WITH OSTEOSARCOMA.