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Mammalian hearing loss is typically permanent due to the inability to replace damaged cochlear hair cells. However, the neonatal mice inner ear demonstrates regenerative capacity, with cochlear floor cells proliferating and differentiating into organoids containing new hair cells and supporting cells, yet the governing molecular mechanisms remain poorly understood. Here, we isolated extracellular vesicles (EVs) from inner ear organoids at proliferation and differentiation stages, characterized their EV miRNA profiles through sequencing, and validated findings using public transcriptomic datasets to elucidate miRNA-mediated regulatory mechanisms during inner ear development. Inner ear organoids were successfully developed from cochlear duct cells, expressing otic progenitor marker SOX2 and hair cell marker Myo7A and demonstrating functional mechano-transduction activity through FM1-43 uptake. Small RNA sequencing identified 35 differentially expressed EV miRNAs between developmental stages. Integrated analysis with public transcriptome datasets revealed 18 genes with significant differential expression, leading to identification of three key regulatory genes—Trp53, Ezh2, and Zbtb4—that exhibited dynamic spatiotemporal expression during inner ear maturation. Pathway analysis demonstrated that these genes are associated with DNA Repair, P53, and Wnt/β-Catenin signaling with remarkable cell-type specificity. Our results demonstrate that EV miRNAs are temporally regulated during organoid development, with predominant downregulation during differentiation. These findings provide crucial insights into developmental mechanisms that could optimize organoid-based models and guide EV miRNA-based therapeutic strategies for hearing restoration.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with limited therapeutic options. Although cisplatin is recommended as a first-line treatment, its clinical efficacy is hindered by the rapid development of drug resistance, highlighting the urgent need for effective strategies to overcome cisplatin resistance.MethodsWe established a NEPC mouse allograft model and performed RNA sequencing to identify genes associated with cisplatin resistance. The role of CCL5 in tumor-stromal crosstalk was investigated using immunofluorescence, ELISA assays, co-culture assays, and CCL5 knockout mice. Mechanistic studies were conducted to explore CCL5/CCR5-mediated signaling pathways. The therapeutic efficacy of cisplatin combined with maraviroc, an FDA-approved CCR5 antagonist, was evaluated in vitro using NEPC cell lines and patient-derived organoids, and in vivo using NEPC mouse models.ResultsHere, we identify a tumor-stromal interaction mediated by the CCL5/CCR5 axis that drives cisplatin resistance in NEPC. Cisplatin-induced DNA damage promotes a cGAS-STING–dependent senescence program in cancer-associated fibroblasts (CAFs), resulting in the secretion of CCL5, a key senescence-associated secretory phenotype factor. CCL5 from CAFs binds to CCR5 on tumor cells, promoting the formation of a CCR5/β-arrestin1/p85 complex that activates the PI3K/AKT pathway. This activation enhances DNA repair, protecting tumor cells from cisplatin-induced apoptosis. Pharmacologic inhibition of the CCL5/CCR5 pathway using maraviroc, an FDA-approved CCR5 antagonist, sensitizes NEPC cells to cisplatin treatment and significantly prolongs survival in NEPC mouse models.ConclusionsOur findings identify the CCL5/CCR5 axis as a key mediator of tumor-stromal crosstalk driving cisplatin resistance in NEPC. Mechanistically, CAF-derived CCL5 activates AKT signaling in tumor cells by promoting the formation of the CCR5/β-arrestin1/p85 complex. Targeting this pathway with maraviroc in combination with cisplatin offers a promising therapeutic strategy for overcoming drug resistance in NEPC.Graphical Abstract Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-025-03552-y.

BACKGROUND: Current antibody-drug conjugates (ADCs) face limitations due to a lack of tumor-selective targets, inefficient internalization, and challenges in reaching tumors in challenging sites, ultimately limiting their therapeutic efficacy. We developed and characterized V66-exatecan, a novel ADC composed of V66, a humanized antibody with high affinity for extracellular DNA (exDNA), conjugated to exatecan via a cleavable linker. This ADC employs a dual-targeting mechanism based on exDNA and ENT2 transporter expression to enhance nuclear drug delivery and tumor specificity. This study evaluates its anti-tumor activity, mechanism of action, ability to treat challenging tumors, and safety profile. METHODS: To validate tumor selectivity, V66 or a control antibody were conjugated to a fluorescent tag and injected intravenously into tumor-bearing mice; biodistribution analysis demonstrated selective accumulation in tumors and nuclear localization within tumor cells. V66 was then conjugated to exatecan via a cleavable linker. In vitro assays across diverse cancer cell lines assessed cytotoxicity, DNA damage response (DDR) activation, and TOP1 degradation. In vivo efficacy was evaluated in xenograft models of triple-negative breast cancer (TNBC) and BRCA1/2-deficient tumors, including intracranial medulloblastoma. These models were used to assess tumor growth inhibition, survival benefit, and blood-brain barrier (BBB) permeability. Toxicity was assessed through a dose-escalation study, with analysis of hematologic parameters, histopathology of major organs, and liver and kidney function tests (ALT, AST, BUN, total protein) following short- and long-term treatment. RESULTS: V66-exatecan demonstrated potent anti-tumor activity in multiple cancer cell lines but not on healthy mouse primary fibroblasts, with EC50 values in the low nanomolar range. It induced robust DDR signaling, TOP1 degradation, and bystander killing effects. BRCA1/2-deficient models exhibited enhanced penetration and sensitivity, with up to 17-fold lower EC50 compared to BRCA-proficient controls. In vivo, V66-exatecan significantly inhibited tumor growth and extended survival in both TNBC and BRCA-mutant CNS tumors, including complete regressions and prolonged median survival in BRCA2-deficient models. Toxicology studies revealed no significant hematologic, renal, hepatic, or bone marrow toxicity, even at high or repeated doses. CONCLUSIONS: V66-exatecan represents a next-generation of ADCs that overcomes key limitations of traditional platforms by exploiting exDNA-driven tumor selectivity and ENT2-mediated nuclear delivery. It demonstrates broad therapeutic efficacy and a favorable safety profile, supporting its potential for treating DDR-deficient and hard-to-reach tumors. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-025-02462-z.