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Archive > Volume 33 Issue 8 > 2026,33(8):822-829. DOI:10.3872/j.issn.1007-385X.2026.08.002 Prev Next
Basic Research
Effects and underlying mechanisms of HSP90β-targeting monoclonal antibody 2G1 on osteosarcoma stem-like characteristics of MG-63 sphere cells
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Abstract:
Objective: To investigate the effects of the HSP90β-targeting monoclonal antibody 2G1 on the biological characteristics of MG-63 sphere cells, an osteosarcoma stem cell (OSC)-enriched model, and to explore its association with changes in Wnt signaling-related proteins. Methods: MG-63 sphere cells were generated from the human osteosarcoma cell line MG-63 by serum-free suspension culture. The target antigen recognized by monoclonal antibody 2G1 was identified by immunofluorescence co-localization and co-immunoprecipitation assays. Flow cytometry was used to determine the expression of cell surface HSP90β in parental MG-63 cells and MG-63 sphere cells. Methylcellulose sphere formation, Transwell migration, and invasion assays were performed to evaluate the effects of 2G1 on the sphere-forming, migratory, and invasive capacities of MG-63 sphere cells. A nude mouse subcutaneous xenograft model was established to assess the effect of 2G1 on the tumorigenic potential of MG-63 sphere cells in vivo. Transcriptome sequencing, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Western blotting were further performed to investigate alterations in signaling pathways and the expression of proteins associated with stem-like characteristics, epithelial-mesenchymal transition (EMT)-related markers, and the Wnt/β-catenin signaling pathway following 2G1 treatment. Results: Immunofluorescence co-localization and co-immunoprecipitation demonstrated that HSP90β was the target antigen recognized by monoclonal antibody 2G1. Flow cytometric analysis showed that the proportion of HSP90β-positive cells on the cell surface was significantly higher in MG-63 sphere cells than in parental MG-63 cells [(20.77 ± 1.29)% vs (2.87 ± 0.33)%, P < 0.001]. Compared with the control group, 2G1 treatment significantly reduced the number of spheres formed (82.67 ± 4.70 vs 42.67 ± 4.00), migrated cells (117.00 ± 5.57 vs 78.33 ± 5.03), and invaded cells (130.70 ± 5.03 vs 89.33 ± 5.03) (all P < 0.001). In the nude mouse xenograft model, 2G1 treatment suppressed tumor growth, with tumor weight and tumor volume inhibition rates of (31.83 ± 16.36)% and (22.00 ± 15.48)% , respectively. Transcriptome sequencing identified 698 differentially expressed genes, including 329 upregulated and 369 downregulated genes. KEGG enrichment analysis indicated enrichment of differentially expressed genes in the Wnt signaling pathway. Western blotting analysis further demonstrated that the expression levels of SOX2, OCT4, N-cadherin, phosphorylated β-catenin (p-β-catenin), and c-Myc were significantly decreased, whereas E-cadherin expression was significantly increased following 2G1 treatment (all P < 0.001). Conclusion: Cell surface HSP90β is highly expressed in MG-63 sphere cells. Targeting HSP90β with monoclonal antibody 2G1 suppresses the sphere-forming, migratory, invasive, and tumorigenic capacities of MG-63 sphere cells and alters the expression levels of stem cell markers, EMT-related markers, and Wnt/β?catenin signaling pathway proteins. These findings suggest that HSP90β may contribute to the regulation of osteosarcoma stem-like characteristics in MG-63 sphere cells and may represent a potential therapeutic target for osteosarcoma.
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