Thyroid cancer cell-derived ectosomes induce macrophage differentiation and remodel the tumor microenvironment

Introduction: Papillary thyroid carcinoma generally features an indolent course, but a subset undergoes aggressive dedifferentiation into anaplastic variants. This malignant progression is heavily influenced by dynamic reciprocal crosstalk within the tumor microenvironment mediated by tumor-derived extracellular vesicles (EVs). Here, we investigated the cargo profiles of size-fractionated ectosomes derived from three thyroid cancer cell lines [BCPAP (papillary), CAL-62 (anaplastic), and 8505C (anaplastic)] and evaluated their capacity to reprogram THP-1 macrophages toward a pro-tumoral state. Methods: EVs were isolated from cell culture supernatants via differential centrifugation at 2,800 × g, 10,000 × g (large microvesicles/oncosomes), and 100,000 × g (smallest microvesicles). Structural lineage markers and immunomodulatory molecules (CD80, CD163, TGF-, GDF15), and HMGB1 were characterized using Western blot, qRT-PCR, flow cytometry, and confocal microscopy. Recipient THP-1 cells were treated with distinct EV fractions to assess activation and M2-like polarization. Results: BCPAP cells yielded the lowest quantity and smallest size EVs which lacked significant immunomodulatory proteins. Conversely, aggressive anaplastic lines (CAL-62, 8505C) shed larger MVs reminiscent of large oncosomes, enriched in CD163 and TGF-. While HMGB1 protein was undetectable across EVs from different cell lines—ruling out necrotic debris contamination—HMGB1 mRNA transcripts were selectively packaged inside the smallest vesicle fractions. Functionally, CAL-62 EVs strongly activated and polarized THP-1 cells into an immunosuppressive M2 phenotype (upregulated CD163 and TGF and down-regulated CD80). Remarkably, although GDF15 protein was absent in all native EVs, CAL-62 EVs induced robust de novo expression of GDF15 in recipient THP-1 macrophages. Conclusions: Our findings demonstrate a distinct decoupling between stemness-associated structural traits and immunosuppressive cargo. Aggressive anaplastic thyroid cancer cells leverage large ectosomes to actively deliver transcriptomic blueprints and reprogram microenvironmental macrophages into autonomous sources of immunosuppressive factors.