Integrating single-cell multi-omics for precision radiotherapy stratification in hypopharyngeal squamous cell carcinoma

Background: Hypopharyngeal squamous cell carcinoma (HPSCC) carries the worst prognosis among head-and-neck cancers, with a 5-year overall survival of only 30%-35%. Despite ionizing radiation as the cornerstone of organ-preserving management, locoregional recurrence affects approximately half of patients. The molecular radiobiology of HPSCC-encompassing DNA damage response (DDR), hypoxia-CAIX signaling, and cancer-associated fibroblast (CAF) remodeling-has not been characterized at single-cell resolution or integrated into a validated precision radiotherapy prognostic model. Methods: GBD 2021 epidemiology data were analyzed. scRNA-seq datasets from treatment-naïve Hypopharyngeal squamous cell carcinoma (GSE234933; cross-validation: GSE185206, GSE164690) were processed with Seurat and hdWGCNA. Additional hdWGCNA analyses were applied to the cancer-associated fibroblast and macrophage compartments. LASSO-penalized Cox regression of TCGA-HNSC Hypopharyngeal carcinoma bulk RNA-seq yielded a 9-gene prognostic signature, independently validated in GSE65858. A nomogram was constructed and evaluated by decision curve analysis. CIBERSORT quantified immune microenvironment composition, and qRT-PCR validated signature genes in HPC cell lines under 6 Gy irradiation. Results: hdWGCNA resolved 22 malignant epithelial modules; M1 (DDR/ATM), M6 (hypoxia-CAIX-LDHA), and M22 (POLQ-SLFN11) showed dominant radiation-response enrichment. Additional hdWGCNA analyses identified Module C3 (CAF-stromal) and Mac2 (immunosuppressive macrophage). LASSO-Cox regression yielded the 9-gene signature (ATM, BRCA1, HIF1A, CA9, LDHA, TP53, SLFN11, POLQ, CDKN1A); bootstrap-corrected AUCs of 0.824/0.793/0.761 (1-/3-/5-year) in TCGA-HNSC were independently reproduced in GSE65858. High-risk patients showed an immunologically cold microenvironment, and qRT-PCR confirmed differential radiation-driven signature gene activation in contrasting-radiosensitivity HPC cell lines. Conclusion: This multi-omics study establishes a validated 9-gene precision radiotherapy prognostic signature for HPSCC, integrating single-cell-resolved mechanistic programs across the hypoxia-CAIX axis, the DDR-ATM-BRCA1 axis, and the POLQ-mediated alternative end-joining radioresistance axis as high-priority theranostic targets. Prospective multicenter clinical validation is mandated before nomogram deployment.