Abstract 6585: Distinct small-EV proteomic landscape mediated by annexin A2 alters recipient cell signaling in triple negative breast cancer metastasis

Cancer metastasis is promoted through interactions between tumor-derived extracellular vesicles (EVs) and recipient cells in distant organs. The lungs are frequently affected as secondary organs in metastatic triple-negative breast cancer (TNBC), occurring in approximately 20-30% of cases. A membrane-associated protein, Annexin A2 (AnxA2), is significantly upregulated in TNBC tissues and EVs, leading to poor outcomes in patients. It is among the most identified EV proteins and recruits microRNAs and proteins into the EVs. Previous reports from the lab have shown a direct correlation between AnxA2 levels in small-EVs (sEVs) and metastasis in the lungs and brain in vivo. This study aims to examine how AnxA2 contributes to the promotion of TNBC lung metastasis mediated by sEVs. MDA-MB-4175 (LM2) lung metastatic TNBC cells were used to downregulate AnxA2 protein, and the metastatic potential was assessed through invasion, migration, and proliferation assays. Downregulation of AnxA2 significantly reduced invasive, migratory, and proliferative potential, as well as decreased plasmin-generating abilities. An optimized differential ultracentrifugation and filtration method was used to isolate sEVs, which were characterized according to MISEV2023 guidelines. sEVs obtained from conditioned cell culture media ranged in size from 30-300 nm and exhibited a distinct double membrane under cryo-electron microscopy. Biological analysis revealed that the sEVs were positive for ESCRT and heat shock proteins, as well as CD9 and CD81 tetraspanins. Following characterization, the sEVs were subjected to quantitative proteomics to assess their protein cargo. Additionally, the uptake by recipient cells and downstream signaling alterations were analyzed. A unique sEV protein landscape was observed, which differentially affected biological processes such as tumor cell viability and proliferation, and the migration of fibroblasts and tumor cells. Altered uptake by recipient non-metastatic TNBC cells and lung fibroblasts was revealed, leading to changes in downstream functional processes. Decreasing AnxA2 expression in TNBC cells diminishes their ability to metastasize by modifying the sEV proteome, thereby influencing interactions with recipient cells at both local and distant sites. These findings uncover a novel role for AnxA2 in promoting TNBC metastasis through sEVs. [This research is supported by the NCI of NIH under Award Number R01CA220273 (JKV), awarded to Dr. Jamboor K. Vishwanatha and partially supported by Cancer Prevention and Research Institute of Texas grant (Award#: RP210046).] Citation Format: Rucha Trivedi, Jamboor K. Vishwanatha. Distinct small-EV proteomic landscape mediated by annexin A2 alters recipient cell signaling in triple negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6585.