CCL5-Mediated Immune Interactions Drive Osteosarcoma Progression: Insights from Mendelian Randomization, Single-Cell Analysis, and Functional Validation

Jihao Chen,1,* Yinyan Shao,2,* Junhua Guo,1 Keke Hu,1 Xuefei Yang,1 Ting Huang,1 Heran Zhou1 1Department of Oncology, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310007, People’s Republic of China; 2Department of General Medicine, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310007, People’s Republic of China*These authors contributed equally to this workCorrespondence: Heran Zhou, Department of Oncology, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, No. 453 Stadium Road, Xihu District, Hangzhou, Zhejiang Province, 310007, People’s Republic of China, Email [email protected] Ting Huang, Department of Oncology, Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University, No. 453 Stadium Road, Xihu District, Hangzhou, Zhejiang Province, 310007, People’s Republic of China, Email [email protected]: Osteosarcoma is a highly aggressive bone malignancy with a complex immune microenvironment. Chronic inflammation and immune cell dysregulation are known to contribute to tumor progression, yet the causal relationships remain largely unclear.Methods: This study integrated Mendelian Randomization (MR), single-cell RNA sequencing (scRNA-seq), bulk transcriptomic analysis, and functional experiments to explore the role of inflammatory cytokines and immune cells in osteosarcoma. MR analyses were performed to identify cytokines and immune cell traits causally related to osteosarcoma risk, followed by scRNA-seq to profile CCL5 expression across cell types. Immune cell infiltration and its association with CCL5 expression were analyzed using the TARGET dataset. Functional assays including qPCR, CCK-8, EdU, and colony formation validated the biological effects of CCL5.Results: MR identified CCL5 as a pro-tumorigenic cytokine with a significant causal association with osteosarcoma (OR > 15). scRNA-seq revealed that monocytes/macrophages and fibroblasts were major CCL5-producing cells. Four immune traits were causally linked to osteosarcoma, including CD86 expression on monocytes and CD127 expression on CD4+ T cells (risk factors), and CD8dim T cell and terminally differentiated CD4+ T cell counts (protective factors). CCL5 expression positively correlated with infiltration of CD8+ T cells, Tregs, and M1 macrophages, and was associated with advanced tumor stage. Co-culture assays confirmed that tumor-associated macrophages with high CCL5 expression enhanced osteosarcoma cell proliferation and colony formation.Conclusion: CCL5 plays a central role in shaping the immune landscape of osteosarcoma and promoting tumor progression. Its spatial association with immune markers such as CD127 and CD86 further supports its regulatory role. These findings provide mechanistic insights and highlight CCL5 as a promising biomarker and immunotherapeutic target in osteosarcoma.Keywords: osteosarcoma, immune cells, cytokines, CCL5, macrophage