Radiotherapy Reconstitutes the Collapsed miRNome of Progressive Neuroblastoma and Promotes a Favorable Tumor Evolutionary Shift

Background: Progressive disease (PD) remains the dominant barrier to cure in high-risk neuroblastoma (HR-NB), driven in part by profound post-transcriptional deregulation that fuels clonal evolution, stemness, immune evasion, and therapy resistance. In this study, we define the molecular architecture of PD-associated microRNA (miR) collapse and demonstrate that radiotherapy (RT) acts as a potent post-transcriptional reprogrammer capable of reversing this trajectory. Methods: miRNome profiling was performed in HR-NB tumors from patients during diagnosis (Dx), intensive multi-modal clinical therapy (IMCT) defiant PD, or residual disease after incorporating RT with IMCT (RD-RT). miRs’ expression profiles were validated with selective miR-320a, miR-423, miR-483-3p, miR-122-5p, miR-3184-5p, miR-21-5p qPCR. RT-influenced changes in cell-identity were investigated by assessing histomorphological alterations (H&E) and multiplex immunofluorescence (mIF) for Ki-67, PD-L1, SOX2, CD44 or p53. Results: IMCT-defying PD displayed a loss of 167 miRs, and none upregulated, corresponding an evolutionary fingerprint for pathways governing cancer stem cell (CSC) maintenance, epithelial to mesenchymal transition (EMT), metabolic rewiring, proliferative fitness, and immune suppression. Strikingly, adding RT reversed 146 of these miRs and induced additional 179 RT-specific miRs, restoring regulatory networks governing apoptosis, p53-mediated DNA-damage response, differentiation/mesenchymal to epithelial transition (MET), and immune visibility. Individual miR-qPCR analysis of miR-320a, miR-483-3p, miR-3184-5p miR-21-5p, and miR-122-5p validated and corroborated the array expression outcomes. Histologic and mIF remarkably substantiated these miR signatures, showing that RT transforms poorly differentiated, plastic, PD-L1+/SOX2+/CD44+ CSC-like PD tumors into differentiated, lineage-stabilized, immune-permissive states characterized by neuropil restoration, ganglioneuroma-like maturation, marginal yet measurable loss of SOX2/CD44, suppression of PD-L1, complete collapse of Ki-67+ proliferative compartments, and significant induction of p53. Conclusion: Collectively, our findings indicated that RT could be a global regulator of tumor state transitions, reinstating miR-mediated governance lost during PD evolution and redirecting tumor fate toward regression. Our results provide proof-of-concept evidence that RT delivers powerful molecular benefits beyond local control, introducing a conceptual framework for miR-guided RT personalization, biomarker development, and rational combination strategies to improve outcomes in HR-NB.