Ectosomes from thyroid cancer cells induce macrophage differentiation and remodel the tumor microenvironment.

Background: Ectosomes are crucial for cell communication and molecule transport, with their heterogeneous content varying across tumors. Thyroid cancer, predominantly the well-differentiated papillary histotype (PTC), can progress to aggressive, undifferentiated anaplastic forms, which promote immunosuppression through EV release. Aim: This study aimed to determine if Extracellular Vesicles (EVs) and Large Oncosomes (LO) released from different thyroid cancer cell lines correlates with aggressiveness, and if their content can induce M1/M2 monocyte polarization, specifically focusing on immunosuppressive M2. Methods: Three thyroid cancer cell lines (BCPAP, FTC 133, CAL 62) were used. LOs were isolated via centrifugation (10000 g 30 min) and smaller EVs via ultracentrifugation (100000 g 90 min) from cell culture supernatants. Healthy donor monocytes were isolated, cultured, and treated with LO from CAL 62 cells. Immunostaining (CD163, CD80, CD68), Western blot, and qPCR characterized EV content and monocyte responses. Results: results showed the aggressive anaplastic CAL 62 cell line released abundant LOs and EVs with diverse content, unlike BCPAP and FTC 133 cells which primarily released EVs. CAL 62 LOs had higher protein content, while smaller EVs contained invasion and EMT-promoting microRNAs (miR9, miR221, miR222). Notably, EVs from CAL 62 and FTC 133 cells contained HMGB1 mRNA, which increased under hypoxia. Crucially, CAL 62-derived LOs and EVs induced the M2 marker CD163 and the immunosuppressive cytokine TGF-beta expression on normal human monocytes. Conclusion: in conclusion, this study demonstrates a direct correlation between the quantity, size, and molecular cargo of EVs/LOs and thyroid cancer cell line aggressiveness. EVs from aggressive anaplastic thyroid cancer cells drive M2 monocyte polarization and suppressive cytokine release, highlighting their critical role in tumor immunosuppression, poor prognosis, and treatment resistance. These findings indicate EVs as promising biomarkers for thyroid cancer progression and aggressiveness.