Abstract PO034: Hypoxia-derived Extracellular Vesicles Promote Triple-Negative Breast Cancer Invasion in Vitro

Abstract The increase of tumor mass and complexity triggers cell invasion, a key step of the metastatic cascade, through hypoxia-derived signalization. The decrease in oxygen levels on the inner tumor mass stimulates the secretion of extracellular vesicles (EV) to the microenvironment, whose cargo may induce an invasive behavior of breast cancer cells. In an attempt to identify key players in hypoxic EV-mediated cell invasion in breast cancer, we are investigating the molecular, cellular and biochemical changes triggered by hypoxic-EVs to triple-negative breast cancer cells (MDA-MB-231). EVs have been isolated from conditioned culture media by differential ultracentrifugation and characterized by particle analysis (NTA), transmission electron microscopy (TEM), protein quantification and western blot of EV markers according to the MISEV2018. EV-treated cells were submitted to invasion assays in i) invasion chamber with extracellular matrix-based hydrogel and ii) a fluorescent gelatin-coated surface. Cell lysate and EV samples were probed for matrix metalloproteases by western blot and zymography. Morphological analysis of cells after EV treatment have been conducted after cytoskeleton staining using fluorescent probes. EVs derived from hypoxia and normoxia-cultured MDA-MB-231 are similarly sized and enriched with CD63, FLOT-1 and ALIX, although hypoxic-EVs have displayed higher protein concentration and particle concentration. Hypoxic EVs promoted cell invasion in an extracellular matrix-based hydrogel after 6h and 16h under normal oxygen conditions. Gelatin degradation has also been observed after EV treatment in normoxia, with results akin to degradation found in cells under hypoxia. While protease activity has been detected in EVs via zymography, we have yet to confirm the role of matrix metalloproteases in EV-related cell invasion. While little morphological changes have been observed upon EV treatment in gelatin-adhered cells, they acquire an epithelial morphology in uncoated circumstances, thus indicating a key role of adhesion molecules to hypoxia-derived invasive behavior. In conclusion, hypoxia-derived EVs from breast cancer cells generate morphological and physiological changes to tumor cells in regular oxygen levels, leading to an invasive behavior in collagen-based matrixes. Citation Format: Bianca Cruz Pachane, Wanessa Fernanda Altei, Heloisa Sobreiro Selistre-de-Araujo. Hypoxia-derived extracellular vesicles promote triple-negative breast cancer invasion in vitro [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO034.