Single-cell transcriptomics reveals glycolytic heterogeneity and identifies STC2 as a key regulator of metabolic reprogramming in osteosarcoma.

Osteosarcoma (OS) exhibits profound metabolic reprogramming, yet the specific cellular subpopulations and regulatory networks governing glycolysis within the tumor microenvironment remain elusive. We integrated single-cell RNA sequencing (scRNA-seq) data from 12 OS samples with bulk transcriptomics and high-dimensional Weighted Gene Co-expression Network Analysis (hdWGCNA). The identified core metabolic regulator was comprehensively validated in vitro using functional assays and Seahorse extracellular flux analysis in MG-63 and U2OS cells. Our single-cell analysis revealed marked metabolic heterogeneity, identifying a dedifferentiated "Chondroblastic" subpopulation characterized by high glycolytic flux and stem-like features. Multi-omics screening pinpointed Stanniocalcin-2 (STC2) as a core prognostic regulator of this phenotype. In vitro validation demonstrated that STC2 knockdown significantly suppressed OS cell proliferation, invasion, and the expression of key glycolytic enzymes (PGK1, LDHA, and GLUT1). Crucially, extracellular flux analysis revealed that STC2 depletion induced a profound metabolic shift, substantially attenuating glycolysis (decreased ECAR) while concomitantly enhancing mitochondrial oxidative phosphorylation (increased OCR). We present a high-resolution single-cell metabolic map of OS, identifying STC2 as a critical regulator of glycolysis-associated tumor progression. Targeting STC2 effectively reverses metabolic reprogramming-shifting energy metabolism from glycolysis toward oxidative phosphorylation-offering a promising therapeutic strategy for osteosarcoma.