Targeting EGFR-TKI resistance in lung cancer: Role of miR-5193/miR-149-5p loaded NK-EVs and Carboplatin combination

Lung cancer remains the leading cause of cancer-related deaths, and there is an urgent need for innovative therapies. MicroRNA (miRNA)-based gene therapy has shown promise, but efficient delivery systems are required for its success. This study investigates the use of extracellular vehicles (EVs) secreted by natural killer (NK) cells as delivery systems for miRNAs targeting PD-L1/PD-1 immune checkpoint and FOXM1, in combination with Carboplatin, to enhance anticancer efficacy in lung cancer models. NK-EVs were isolated from NK92-MI cells and characterized using nanoparticle tracking analysis (NTA), proteomics and Western blotting, confirming their exosomal characteristics. Gene ontology profiling and RNA-seq identified highly expressed miRNAs such as miR-5193 and miR-149-5p, which were loaded into NK-EVs via electroporation. Agarose gel electrophoresis confirmed their entrapment and Quickdrop spectrophotometer was used to estimate the quantity. In vitro, miRNA-loaded NK-EVs demonstrated significant cytotoxicity against Osimertinib-resistant PDX (TM0019, Jackson Labs) and H1975R (with L858R mutations) lung cancer cells, with approximately 1.2 to 1.6-fold (p < 0.01) decrease in cell viability compared to NK-EVs alone. In vivo, the combination of miRNA-loaded NK-EVs and Carboplatin significantly reduced tumor volumes (3.5 to 4-fold, p < 0.001) in PDX and H1975R xenograft models, with the most pronounced effect observed in combination therapies. Western blot analysis showed downregulation of tumor-associated markers: PD-1/PD-L1, FOXM1, Survivin, NF-κB and others vs untreated group, p < 0.001) suggesting immune checkpoint inhibition, apoptosis and anti-inflammatory activity. These findings highlight the potential of NK-EVs as effective carriers for miRNAs in combination with chemotherapy, offering a promising therapeutic strategy for NSCLC with EGFR mutations.