Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

Extracellular vesicles (EVs) are critical mediators of intercellular communication within the tumour microenvironment and play a significant role in drug resistance. We aimed to investigate the mechanisms underlying gemcitabine (GCB) resistance in bladder cancer. GCB-resistant bladder cancer cells exhibited dysregulation of nucleoside-metabolizing enzymes and transporters. Characterization of EV subpopulations derived from GCB-resistant cells revealed their ability to transfer drug-resistant phenotypes to naïve cancer cells by modulating intracellular levels of nucleoside metabolic proteins and transporters. Proteomic and transcriptomic analyses identified the histone protein H3.2 and its corresponding transcript, H3C14, as key regulators in the transmission of GCB resistance. Notably, H3C14 overexpression in resistant cells restored GCB sensitivity, whereas its knockdown induced GCB resistance. Rab27A-mediated biogenesis and secretion emerged as a crucial mechanism regulating EV release and H3C14 excretion in GCB-resistant cells. A specific EV subpopulation enriched in CD147 and LAMB1-referred to as Excretion EVs-carried H3.2 (H3C14) but did not induce GCB resistance in recipient cells, suggesting their primary role in eliminating proteins associated with tumour progression and drug resistance. These findings highlight the role of EV-mediated H3C14 excretion in regulating GCB resistance and suggest potential therapeutic strategies targeting EV pathways to overcome drug resistance in bladder cancer.