A novel pathway for stemness propagation and chemoresistance in non-small cell lung cancer via phosphorylated PKM2-loaded small extracellular vesicles.

Rationale: Non-small cell lung cancer (NSCLC) is a predominant cause of cancer-related mortality, with its progression and treatment resistance significantly influenced by cancer stem cells (CSCs) and their complex intercellular communication mechanisms. Small extracellular vesicles (sEVs) have emerged as pivotal mediators of intercellular signaling, affecting tumor microenvironment modulation and therapeutic resistance. This study investigates the role of CSC-derived sEVs in transmitting stemness traits through the selective sorting of pyruvate kinase M2 phosphorylated at the Y105 site (pY105-PKM2), mediated by the adaptor protein IQGAP1, which supports CSC maintenance and drug resistance in NSCLC. Methods: In vitro and in vivo experiments, including proteomic and transcriptomic analyses, were conducted to identify key regulators of sEV-mediated signaling. Immunoprecipitation, proximity ligation assays, and immunofluorescence were used to examine the role of IQGAP1 in the sorting of pY105-PKM2 into sEVs. Functional assays, including sphere formation, chemoresistance tests, metabolic assessments, and cell cycle analysis, were conducted to evaluate the effects of sEV-mediated delivery of pY105-PKM2 on recipient cells. Additionally, immunohistochemistry and survival analysis were performed on tumor samples from NSCLC patients to establish clinical correlations. Results: We unveiled a novel mechanism by which CSC-derived sEVs transmit stemness traits to replenish the CSC pool in NSCLC. CSC-derived sEVs were enriched with pY105-PKM2, correlating with enhanced stemness, chemoresistance, and poor clinical outcomes. Mechanistically, IQGAP1 was identified as an adaptor facilitating the selective sorting of pY105-PKM2 into sEVs through interactions with the ESCRT component TSG101. Recipient cells treated with CSC-derived sEVs exhibited metabolic reprogramming, slower cell cycle progression, and enhanced chemoresistance. The synergistic role of IQGAP1 and pY105-PKM2 was confirmed, highlighting their critical contributions to CSC maintenance and malignant progression. Conclusion: This study highlights the critical role of CSC-derived sEVs in NSCLC progression and therapy resistance through the IQGAP1-mediated selective sorting of pY105-PKM2. By uncovering this novel pathway, our findings provide valuable insights into CSC pool replenishment and therapeutic resistance mechanisms in NSCLC, identifying IQGAP1 and pY105-PKM2 as promising therapeutic targets for mitigating CSC-driven malignancy and enhancing treatment efficacy.