(629d) Extracellular Vesicle Secretion Drives Radiation-Induced Bystander Effects in Breast Cancer

Although primary tumors are typically managed through a combination of surgery, radiotherapy, and chemotherapy, triple negative breast cancer (TNBC) patients experience relatively high rates of recurrence after treatment. The role of intracellular communication in this process is unknown. Recent studies have shown that ionizing radiation (IR) activates several systemic biological responses, which largely depend on interactions between healthy and damaged tissue cells. Radiation-induced bystander effects (RIBE) are secondary effects that develop in non-irradiated cells as a result of communicative signals sent directly by irradiated cells. The diversity of biological consequences associated with RIBE suggests that complex intracellular signals are involved. Therefore, we hypothesized that extracellular vesicles (EVs)—membrane-delimited structures containing all major classes of biomolecules—function as mediators of RIBE, leading to TNBC progression. This study represents a crucial step toward elucidating how microenvironmental changes caused by IR influence cancer recurrence.

We explored the impact of IR on EV secretion in stromal cells. In vitro cells representative of breast tissue were utilized in these experiments. Mouse 3T3 fibroblasts and 3T3-L1 pre-adipocytes were irradiated to a dose of 10 Gy. We used nanoparticle tracking analysis (NTA) and flow cytometry to characterize EV secretion dynamics 24 hours post-irradiation in control and irradiated cells. Furthermore, we evaluated the ability of irradiated stromal cell-derived EVs to alter untreated recipient cells. We visualized fibroblast and pre-adipocyte cytoskeletal dynamics following EV treatment by phalloidin staining of F-actin. We also treated murine 4T1 TNBC cells with isolated EVs and evaluated migration through a transwell assay and proliferation through Ki67 staining. Finally, we utilized mass spectrometry to analyze differentially regulated EV proteins after IR.

Our NTA and flow cytometry results indicate that irradiation enhances vesicular secretion and alters the distribution of vesicle subpopulations. Additionally, our findings suggest the ability of irradiated cells to induce bystander effects through EVs. Treating non-irradiated fibroblasts and pre-adipocytes with EVs from irradiated fibroblasts led to cytoskeletal rearrangement as determined through quantitative analysis of the anisotropy of F-actin fibers. Interestingly, this trend is in agreement with the observance of higher f-actin alignment in directly irradiated cells. Furthermore, EVs from irradiated fibroblasts and pre-adipocytes enhanced the migration and proliferation of TNBC cells. Finally, our mass spectrometry results suggest differential packing of key proteins into EVs after cellular exposure to IR.

Overall, our results establish cell-specific changes arising from interactions between irradiated and non-irradiated cells through EVs, suggesting involvement with local and systemic RIBE. Notably, EVs derived from irradiated fibroblasts resulted in a higher degree of actin fiber alignment in non-irradiated fibroblasts and pre-adipocytes while leading to enhanced cancer cell migratory and proliferative capacity. This work will further our understanding of EV-mediated communication patterns that arise after radiotherapy and will lead to the development of novel therapies for preventing TNBC recurrence.