Integrative bulk and single-cell transcriptomics identifies a HERC5-linked immune-attenuated risk program for pulmonary metastasis/relapse in osteosarcoma

BACKGROUND: Pulmonary metastasis/relapse (PM/R) is a major cause of osteosarcoma treatment failure. We aimed to develop a compact transcriptomic PM/R risk score and characterize associated immune and single-cell programs. METHODS: TARGET-OS bulk RNA-seq was used for discovery and GSE21257 for external validation. Differential expression, multi-algorithm feature selection and LASSO regression were used to construct the score. Clinical associations were assessed by PM/R classification, survival and calibration analyses. Immune signature scoring, weighted gene co-expression network analysis (WGCNA), single-cell projection, in silico virtual HERC5 knockout, PROGENy/DoRothEA, CellChat and exploratory drug-reversal analyses were used for biological interpretation. RESULTS: A 9-gene score comprising HERC5, GPR141, SLC45A4, LCN15, MXI1, UCK2, KRTAP13-2, KCNK6 and TRABD2A showed high apparent discrimination in TARGET-OS (AUC = 0.922). A reduced seven-gene GSE21257 implementation achieved an AUC of 0.728. High-risk tumors were enriched for PM/R and had poorer event-free survival (HR = 3.14, 95% CI, 1.60-6.16) and overall survival (HR = 3.75, 95% CI, 1.60-8.79). High-risk disease showed reduced antigen-presentation, IFN-gamma, T-cell, CD8 T-cell, NK-cell and cytotoxicity signatures. WGCNA identified a protective immune module, while single-cell and in silico virtual-knockout analyses prioritized HERC5 as a candidate immune-associated node linked to antigen-presentation genes. Regulatory and communication analyses suggested reduced JAK-STAT/IRF activity and SPP1/MIF-centered microenvironmental signaling. CONCLUSIONS: This study defines a PM/R-focused transcriptomic risk score and links high-risk osteosarcoma to immune attenuation and a HERC5-associated antigen-presentation program, providing a framework for future validation.