2024-04-162024-04-162024-03-21https://hdl.handle.net/20.500.12503/32607Purpose – Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer poses a high risk of metastasis due to a lack of targeted therapy and reliance on conventional treatment approaches. Among the organs susceptible to metastasis, the lungs are most frequently affected sites, accounting for approximately 60% of cases. Extracellular vesicles (EV) play a crucial role in establishing a pre-metastatic niche (PMN) for tumor cell homing to secondary sites. Identifying the tumor-derived EV-specific cargo that may potentially alter the lung microenvironment could establish an understanding of the molecular targets contributing to lung PMN development and consequently improving the poor TNBC prognosis and diagnosis. TNBC exhibits elevated expression of Annexin A2 (AnxA2), a plasma and endosome membrane-associated protein. The abundant expression of AnxA2 in tumor tissues and EVs derived from TNBC patients is correlated with poor overall survival and poor distant metastasis-free survival. In previous studies, depletion of AnxA2 protein levels in TNBC-derived EVs resulted in an altered PMN establishment, leading to significantly lower metastatic lesions in the lungs and brain in in vivo system. We aim to evaluate the molecular effects mediated by AnxA2 in TNBC-derived EVs and their cargo that aid the PMN formation and identify underlying molecular targets creating a favorable microenvironment by interaction with lung stroma. Methods and Results – To assess the role of AnxA2 in TNBC lung metastasis, we utilized a lung selective metastatic TNBC cell line, MDA MB 4175 (LM2). We used the shRNA-mediated stable knockdown technique to downregulate AnxA2 protein levels in LM2 cells. We then performed expressional analysis as well as its cell surface functionality using a plasmin generation assay. Additionally, we conducted tumor cell functional assays such as proliferation and invasion assays. Decreased AnxA2 protein levels in LM2 cells significantly reduced its plasmin generation, proliferative ability, and invasive potential. Subsequently, we isolated cell derived small EVs using differential ultracentrifugation and characterized them following the MISEV 2018 guidelines. We confirmed the purity, yield, size, presence of EV-enriched proteins (ESCRT, Hsp, CD9, CD81), and absence of negative proteins (Calnexin, GM130). We then subjected the EVs to quantitative proteomic analysis and identified the differentially expressed proteins upon AnxA2 depletion. We further exposed the healthy lung stromal fibroblasts and the local less-aggressive TNBC cells with EVs to assess the differences in local and distant site uptake and cargo transfer mediated by AnxA2. Conclusion – This comprehensive approach will determine the novel EV-associated proteins that act as functional regulators in promoting TNBC metastasis to the lungs. [This work is supported by the NCI R01CA220273 awarded to Dr. Jamboor K. Vishwanatha]enposter