MiR-146a-armed exosomes reverse immunosuppressive networks in NSCLC by dual-targeting of MYD88/STAT3 and PD-L1 axes.

BACKGROUND

MiR-146a has been found to be a tumor suppressor and is downregulated in NSCLC cells. This study investigated the effects of delivering miR-146a via tumor-derived exosomes (TEX-MiR-146a) on the behavior of malignant cells and immunological modulation in NSCLC models.

METHODS

miR-146a expression was evaluated in A-549 and normal MRC-5 lung epithelial cells. Exosomes were isolated from A-549 cells, loaded with a miR-146a mimic, and characterized for size, morphology, surface markers, and loading efficiency. In vitro, the functional effects of TEXmiR-146a were compared to controls using tests for cell survival, apoptosis, migration, invasion, wound healing, colony formation, and gene expression. Co-culture immunological assays, including T cell subset and cytokine profiling, were also performed using patient-derived peripheral blood mononuclear cells (PBMCs).

RESULTS

TEXmiR-146a efficiently loaded miR-146a into cells (60.1 % ± 4.0 efficiency, P < 0.0001). Treatment with TEXmiR-146a (25 μg/ml) significantly reduced the viability of A-549 cells by ∼51 % after 48 h (P < 0.0001). Cell migration and invasion were inhibited by >50 %, with migrating cells reduced to 116.7 ± 7.09 (P < 0.001) and invading cells to 171.7 ± 6.028 (P < 0.001), respectively, compared to controls. Pro-apoptotic genes Bax/Bcl-2 (P = 0.0002) and Caspase-3 were upregulated (P = 0.0003), while metastatic and immunoregulatory genes VEGF-A, MMP-9, STAT3, MYD88, and PD-L1 were downregulated (all P < 0.05). In patient PBMCs, TEXmiR-146a increased the frequency of cytotoxic CD8+ T cells (24.75 ± 1.23 % versus 20.15 ± 1.26 %, P < 0.0001) and decreased Treg cells (2.01 ± 1.22 % versus 4.04 ± 1.72 %, P = 0.015) compared to the TEX group, alongside a significant reduction in TGF-β (P = 0.01).

CONCLUSION

Exosomal delivery of miR-146a effectively restores tumor suppressor functions and reverses immunosuppressive networks in NSCLC by dual-targeting key oncogenic and immunomodulatory axes, presenting a potent cell-free immunotherapeutic strategy.