Small Extracellular Vesicles Orchestrate Cisplatin‐Induced Ototoxicity: Potential Biomarker and Targets Discovery

Cisplatin-induced ototoxicity remains a clinical dilemma with limited mechanistic understanding and no food and drug administration (FDA)-approved therapies. Despite emerging roles of small extracellular vesicles (sEV) in drug ototoxicity, their molecular cargo profiles and causal roles to cisplatin-induced ototoxicity are unexplored. This study systematically investigates sEV derived from cochlear explants treated with cisplatin (Cis-sEV) and controls (Ctrl-sEV) using multi-omics profiling. Through small RNA sequencing, 83 differentially expressed microRNAs (miRNAs) are identified in Cis-sEV compared to Ctrl-sEV. Notably, mmu-miR-34a-5p, mmu-miR-140-5p, mmu-miR-15b-5p, mmu-miR-25-3p, and mmu-miR-339-5p are significantly upregulation in Cis-sEVs. Predicted target pathways of these differentially expressed miRNAs are enriched in apoptosis, inflammation, and cellular damage, indicating their potential involvement in cisplatin-induced cochlear damage. LC-MS/MS analysis reveals 90 upregulated and 150 downregulated proteins in Cis-sEV, with many involved in damage response. Specifically, CLTC, CCT2, ANXA6, and HSPA8 are uniquely upregulated proteins in Cis-sEV, and CLTC and ANXA6 are exclusively co-localized in hair cells (HCs) post-cisplatin exposure, suggesting that Cis-sEV originate primarily from damaged HCs. Moreover, CLTC in sEV may serve as a potential biomarker for cisplatin-induced ototoxicity as verified in both in vitro and in vivo models. This study provides novel insights into the molecular mechanisms of cisplatin-induced ototoxicity and identifies potential biomarker and therapeutic targets.