Integrated RNA sequencing reveals tumor microenvironment heterogeneity and immunosuppressive role of M2 macrophages in osteosarcoma

Abstract Objectives Osteosarcoma, a prevalent malignant bone tumor in pediatric and adolescent populations, is characterized by a markedly diverse tumor microenvironment (TME) that complicates therapeutic intervention. Among the key components of the TME, M2 macrophages contribute to immune suppression, tumor progression, and resistance to therapy. This study aims to explore the immune microenvironment of osteosarcoma and identify key immunosuppressive components using integrated transcriptomic analysis. Methods We integrated scRNA-seq (GSE152048) and bulk RNA-seq (TARGET) data to characterize the osteosarcoma tumor microenvironment. Key analyses included cell clustering and annotation (Seurat), pseudotime trajectory (Monocle 2), cell-cell communication (CellChat), and M2 macrophage deconvolution (CIBERSORT, BayesPrism). Gene modules were identified via WGCNA, and drug sensitivity was predicted using oncoPredict. Key genes were validated by qRT-PCR and Western blot. Results Our integrated analysis revealed that M2 macrophage infiltration was significantly associated with an immunosuppressive tumor microenvironment in osteosarcoma. Weighted gene co-expression network analysis (WGCNA) identified gene modules highly correlated with M2 macrophage abundance and immune evasion signatures. Reduced M2 infiltration was linked to altered transcriptional patterns and increased predicted sensitivity to Etoposide and Doxorubicin. Two immune-related genes, PTK2B and PTPRC, were significantly upregulated in osteosarcoma samples and validated by RT-qPCR and Western blotting in U2OS cells. Conclusions This study demonstrates that M2 macrophage infiltration is a defining feature of the osteosarcoma immune microenvironment and is associated with immune suppression. Through integrative transcriptomic analysis, PTK2B and PTPRC were identified as key immune-related genes linked to M2 macrophage abundance and patient prognosis. These results establish a transcriptional basis for the immunosuppressive phenotype in osteosarcoma.