miR-221/222 Profiles in Small Extracellular Vesicles Derived from Gemcitabine-Sensitive and -Resistant A549 Cancer Stem-Like Cell Spheroids: An In Vitro Study

Abstract

Background: Gemcitabine is used in the treatment of human non-small cell lung cancer (hNSCLC), yet acquired resistance can limit therapeutic responses. Small extracellular vesicles (sEVs) and microRNAs (miRNAs) have been implicated in chemoresistance-associated intercellular signaling. We profiled the miR-221/222 cluster and resistance-associated genes in cancer stem-like cells (CSLCs) and CSLC-derived sEV preparations from gemcitabine-sensitive and -resistant A549 models. Methods: Gemcitabine-resistant A549 cells and matched cancer stem-like cell (CSLC) and gemcitabine-resistant cancer stem-like cell (Gr-CSLC) populations were generated and characterized by flow cytometry, qRT-PCR, and immunofluorescence. sEV preparations derived from CSLC and Gr-CSLC spheroids were characterized for total protein content, EV-associated tetraspanins, and vesicular morphology. PKH26-associated fluorescence following exposure of A549 cells to labeled sEV preparations was also evaluated. The relative levels of sEV-associated miR-221/222 family members and the resistance-associated genes MDR1, ABCG2, and BCL2 were assessed by qRT-PCR. Results: Gr-CSLCs exhibited enhanced spheroid formation and increased MDR1 expression. Gr-CSLC-derived sEV preparations showed higher total protein content than CSLC-derived preparations. Bead-based flow cytometry demonstrated EV-associated CD9 and CD81 expression, while cryo-TEM revealed small, membrane-bound vesicular structures. PKH26-associated intracellular fluorescence was observed in A549 cells following exposure to labeled sEV preparations. Gr-CSLC-derived sEVs showed higher relative levels of miR-221-3p, miR-222-3p, and miR-222-5p than CSLC-derived sEVs, whereas miR-221-5p showed no significant difference. MDR1 and BCL2 also showed higher relative expression in Gr-CSLC-derived sEVs, whereas ABCG2 showed no significant difference. Conclusions: sEV preparations derived from gemcitabine-resistant Gr-CSLCs exhibited distinct miR-221/222 and resistance-associated RNA profiles compared with those derived from gemcitabine-sensitive CSLCs in this in vitro A549 NSCLC model. These molecular differences should be interpreted as associations with the resistant phenotype rather than evidence of functional transfer of chemoresistance. Further functional studies and validation in additional NSCLC and patient-derived models are required to establish their biological, therapeutic, or biomarker relevance.