Machine Learning–Based Identification and Validation of PYCR1 and PYGM as Prognostic Biomarkers for Osteosarcoma

Background: Osteosarcoma (OS) is a malignant tumor originating in the bones, predominantly affecting children and adolescents, characterized by high aggressiveness and poor prognosis. Identifying new prognostic biomarkers is crucial for improving the diagnosis and treatment of OS. Methods: In this study, we collected gene expression data from 88 OS samples from the UCSC Xena platform and normal tissue expression data from 396 Genotype-Tissue Expression (GTEx) samples. Prognosis-related genes were first screened by univariate Cox regression and then further selected using the Least Absolute Shrinkage and Selection Operator (LASSO) regression. Based on these candidate genes, non-negative matrix factorization (NMF) was used for molecular subtype identification, and the Kaplan-Meier analysis was applied to compare survival among subtypes. Tumor microenvironment and immune cell infiltration analyses were performed to characterize differences between risk groups. In addition, the expression patterns of key genes were validated by quantitative real-time polymerase chain reaction (qRT-PCR), hematoxylin-eosin staining, immunohistochemistry, and immunofluorescence. Results: Pyrroline-5-carboxylate reductase 1 (PYCR1) was consistently upregulated in OS and was associated with poor prognosis. In contrast, glycogen phosphorylase, muscle-associated (PYGM) showed analysis-level-dependent expression patterns: it was downregulated at the bulk transcriptomic and tumor cell levels compared with normal controls, whereas within the OS cohort, relatively higher PYGM expression was observed in the high-risk group. Tumor microenvironment and immune cell infiltration analyses revealed significant immune differences between high- and low-risk groups. Histological and protein-level assays further confirmed the presence and cellular localization of PYCR1 and PYGM in OS tissues. Conclusion: This study systematically identified and validated PYCR1 and PYGM as potential prognostic biomarkers for OS using integrated statistical and machine learning approaches. The PYCR1 showed a consistently tumor-promoting expression pattern, whereas PYGM demonstrated context-dependent expression changes across bulk tissue, risk-stratified tumor samples, and tumor cell lines, highlighting the biological complexity of metabolic biomarkers in OS.