Integrative Bulk, Single-Cell, and Spatial Transcriptomic Analyses Nominate PPARG as a Candidate Senescence-Related...

Osteosarcoma remains challenging in metastatic, recurrent, or therapy-resistant disease. This study aimed to identify senescence-related prognostic genes and characterize their spatial contexts. Paired differential expression analysis was performed in GSE99671 using DESeq2, followed by intersection with the CellAge senescence gene set. Transcriptomic data from the TARGET-OS cohort were obtained from UCSC Xena. Candidate genes were evaluated using univariate Cox regression, Kaplan-Meier analysis, time-dependent receiver operating characteristic analysis, LASSO Cox regression, repeated LASSO analysis, and random survival forest modeling, with clinical covariates incorporated into adjusted Cox models. Functional enrichment, immune microenvironment analysis, single-cell transcriptomics, SP_BS3 spatial transcriptomics, GSE36001 expression validation, and qRT-PCR and Western blot validation in osteosarcoma 143B cells and osteoblast cells were used to characterize it. In GSE99671, 2,248 genes were differentially expressed with adjusted P < 0.05, and their intersection with the 866 CellAge genes yielded 105 senescence-related differentially expressed genes. In TARGET-OS, lower PPARG expression was associated with a higher mortality risk (univariate HR = 0.603, 95% CI = 0.454-0.802, P = 0.000494; adjusted HR = 0.224, 95% CI = 0.085-0.589, P = 0.00241). Adding PPARG to the clinical model increased the C-index from 0.707 to 0.829. PPARG was downregulated in both GSE99671 and GSE36001, and qRT-PCR and Western blotting confirmed lower PPARG mRNA and protein expression in osteosarcoma 143B cells than in osteoblast cells. Single-cell analysis localized PPARG to endothelial cells, pericytes, macrophages/monocytes, and tumor-associated stromal cells. Spatial transcriptomic analysis showed weak but significant positive correlations between PPARG expression and the CellAge senescence, endothelial-related, and pericyte-related scores. These findings nominate PPARG as a candidate CellAge-derived prognostic biomarker associated with unfavorable survival and vascular microenvironmental features in osteosarcoma and support investigation of its potential relevance to risk stratification and the senescence-related tumor microenvironment. GSE36001 provided external expression validation only; independent survival validation was not performed.