Verification of Ferroptosis Subcluster-Associated Genes Related to Osteosarcoma and Exploration of Immune Targeted Therapy

BACKGROUND: Despite tremendous advances in treating osteosarcoma (OS), the survival rates of patients have failed to improve dramatically over the past decades. Ferroptosis, a newly discovered iron-dependent type of regulated cell death, is implicated in tumors, and its features in OS remain unascertained. We designed to determine the involvement of ferroptosis subcluster-related modular genes in OS progression and prognosis. METHODS: The OS-related datasets retrieved from GEO and TARGET database were clustered for identifying molecular subclusters with different ferroptosis-related genes (FRGs) expression patterns. Weighted gene coexpression network analysis (WGCNA) was applied to identify modular genes from FRG subclusters. The least absolute shrinkage and selection operator (LASSO) algorithm and multivariable Cox regression analysis were adopted to develop the prognostic model. Potential mechanisms of development and prognosis in OS were explored by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA). Then, a comprehensive analysis was conducted for immune checkpoint markers and assessment of predictive power to drug response. The protein expression levels of the three ferroptosis subcluster-related modular genes were verified by immunohistochemistry. RESULTS: Two independent subclusters presenting diverse expression profiles of FRGs were obtained, with significantly different survival states. Ferroptosis subcluster-related modular genes were screened with WGCNA, and the GESA results showed that ferroptosis subcluster-related modular genes could affect the cellular energy metabolism, thus influencing the development and prognosis of osteosarcoma. A prognostic model was established by incorporating three ferroptosis subcluster-related modular genes (LRRC1, ACO2, and CTNNBIP1) and a nomogram by integrating clinical features, and they were evaluated for the predictive power on OS prognosis. The 20 immune checkpoint-related genes confirmed the insensitivity to tumor immunotherapy in high-risk patients. IC50s of Axitinib and Cytarabine suggested a higher sensitivity to the targeted drug. Finally, the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry were consistent with bioinformatics analysis. CONCLUSION: Ferroptosis are closely associated with the OS prognosis. The risk-scoring model incorporating three ferroptosis subcluster-related modular genes has shown outstanding advantages in predicting patient prognosis.