Abstract 6588: Characterization of TGFb1-induced ApoEVs and their potential role in epithelial cell plasticity

Abstract Extracellular vesicles (EVs) are membranous particles that carry cargo such as proteins, lipids, and nucleic acids that can be crucial for intercellular communication. Epithelial-mesenchymal transition (EMT) is implicated in tissue remodeling and has been documented in the mammary gland during involution. Transgenic models and clinical studies suggest that both post-lactational and age-related lobular involution may increase susceptibility for breast cancer development. Transforming growth factor-beta 1 (TGFβ1) induces apoptosis of epithelial cells during involution and is also a potent inducer of EMT. TGFβ-induced apoptosis generates a subpopulation of EVs called apoptotic cell-derived EVs (ApoEVs), a potential cell-cell communication avenue that may be crucial for tissue remodeling. Here, we optimized a protocol to generate, isolate, and analyze TGFβ-induced ApoEVs. This may reveal potential EV-mediated signaling in driving EMT, tissue remodeling and cancer susceptibility in the mammary gland. The murine mammary gland epithelial cell line NMuMG was used, as they are known to both die and undergo EMT in response to TGFβ1 stimulation. NMuMG cells were cultured in DMEM with 10% FBS, 10 µg/mL insulin, and 1% antibiotics. Once 90% confluency was reached, growth media was replaced with starvation media consisting of DMEM, 5% EV-depleted FBS, and antibiotics. After 2.5 hours, one plate was treated with 5 ng/mL of TGFβ1 while the other received PBS. The conditioned media (CM) produced was centrifuged at 400g for 20 minutes to remove debris and then ultracentrifuged for 1 hour at 4ºC at a speed of 10, 000g to generate a pellet of large EVs. This was followed by two PBS washes under the same ultracentrifugation conditions. Next, the CM was again ultracentrifuged at a speed of 100, 000g to isolate smaller EVs. Both EV populations were resuspended in 10mM Hepes (pH 7.2) and characterized by western blot, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Western blots for markers of EVs and potential contaminants suggest successful EV isolation. Blotting for apoptotic markers presented results consistent with ApoEVs, which were enriched in the samples obtained by 10, 000g ultracentrifugation. DLS analysis showed that EVs released by TGFβ-treated cells were larger and more abundant compared to those released by untreated cells; TEM analysis confirmed these results. Culturing NMuMG cells with either full CM or EV-depleted CM from either control or TGFβ1-treated cells showed morphological changes consistent with cell plasticity in response to EVs released by TGFβ1-treated cells. Our findings suggest that EVs produced during TGFβ1-induced apoptosis of NMuMG cells contain cargo capable of modulating epithelial phenotype and may aid directly or indirectly in tissue remodeling. Additional biological studies on target cells are necessary to confirm our results. Citation Format: Daxime F. Génier, Sarai A. Gomez Perez, Alicia Viloria-Petit. Characterization of TGFb1-induced ApoEVs and their potential role in epithelial cell plasticity [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 6588.