Improving radiotherapy efficacy in melanoma using engineered tumor-derived extracellular vesicles

Introduction: Melanoma poses a significant challenge due to its aggressive nature and resistance to conventional therapies. Standard treatment approaches often result in relapse among advanced-stage patients, prompting the search for novel strategies. Recent progress in engineering Extracellular Vesicles (EVs) to carry anti-tumoral molecules have shown promising prospects in cancer treatment. Additionally, RNA-based therapeutics aimed at targeting key resistance pathways have demonstrated encouraging perspectives as adjuvants. We recently showed the potential of melanoma-derived EVs enriched with miR-195-5p, a tumor suppressor miRNA, to inhibit tumor growth and sensitize cells to targeted therapy via miR-195-5p/BCL2-L1 axis. Given the predicted regulatory role of miR-195-5p in DNA damage response (DDR)-related genes, we aimed to investigate the effect of miR-195-5p-loaded EVs in melanoma spheroids growth and response to radiotherapy (RT), which is commonly used in unresectable cases and as palliative care for melanoma patients. Methods: EVs were isolated from primary (A375 and WM-1366) and metastatic (SKMel-28, SKMel-147, and UACC-62) melanoma cell conditioned media using differential ultracentrifugation followed by size exclusion chromatography. Nanoparticle Tracking Analysis, cryo-electron microscopy, and western blotting for common markers (CD63 and CD9) were conducted following MISEV2023 guidelines. MiR-195-5p was loaded into isolated vesicles via electroporation, confirmed by RT-qPCR post RNAse A treatment. 3D tumor spheroids were obtained 72 hours post-plating (1,000 cells/well) in 96 multiwell plates coated with a 1% agarose layer. Three-day-old spheroids were submitted to fractionated RT for 5 days (5 x 3Gy), with EVs added 1 hour before the last three doses. Results: Melanoma spheroids exhibited high resistance to fractionated radiotherapy (RT), accompanied by increased expression of DNA damage repair and stemness genes. EVs electroporation resulted in increased intravesicular miR-195-5p levels (by 100x). Although miR-195-loaded vesicles were only taken-up by cells in the inner surface of the spheroids, they could delivery this miRNA, inducing anti-proliferative effects. While combined RT/miR-195 EVs treatment did not significantly impact cell death or DNA repair capacity, it did result in decreased tumor growth upon repopulation challenge. Additionally, the observed upregulation of miR-195 was associated with a suppression of RT-induced resistance genes (ALDH1, ATM, OCT4, SOX2, PDL-1, LINC00473, LINC00511), possibly through miR-195/ZBTB37 axis. Conclusion: MiR-195 EVs demonstrate potential as an adjuvant therapy to enhance radiotherapy efficacy in melanoma.  Moreover, further enhancements in their capacity to penetrate tumor tissues could amplify their therapeutic impact. Funding: FAPESP (2021/13681-2).