目的：以MG-63球形细胞作为骨肉瘤干细胞（OSC）富集模型，探讨靶向细胞膜表面HSP90β的单抗2G1对其生物学特征的影响及其与Wnt信号相关蛋白变化的关系。方法：采用无血清悬浮培养人骨肉瘤MG-63细胞获得MG-63球形细胞。通过免疫荧光共定位和免疫共沉淀实验鉴定单克隆抗体2G1识别的靶抗原；采用流式细胞术检测MG-63亲本细胞和MG-63球形细胞膜表面HSP90β的表达水平；通过甲基纤维素成球实验、Transwell迁移和侵袭实验评价单抗2G1对MG-63球形细胞成球、迁移和侵袭能力的影响；采用裸鼠皮下移植瘤模型观察单抗2G1对MG-63球形细胞体内成瘤能力的影响；进一步结合转录组测序、京都基因与基因组百科全书（KEGG）通路富集分析及WB检测，分析单抗2G1处理后相关分子通路及干细胞特性、上皮-间充质转化（EMT）、Wnt/β-catenin信号通路相关蛋白表达变化。结果：免疫荧光共定位和免疫共沉淀结果显示，单抗2G1识别的靶抗原为HSP90β。流式细胞术结果显示，MG-63球形细胞膜表面HSP90β阳性表达细胞比例为（20.77 ± 1.29）%，显著高于MG-63亲本细胞的（2.87 ± 0.33）%（P < 0.001）。与对照组相比，单抗2G1处理后MG-63球形细胞成球数由82.67 ± 4.70降至42.67 ± 4.00，迁移细胞数由117.00 ± 5.57降至78.33 ± 5.03，侵袭细胞数由130.70 ± 5.03降至89.33 ± 5.03（均P < 0.001）。裸鼠移植瘤实验显示，单抗2G1组移植瘤生长受到抑制，瘤质量抑制率和瘤体积抑制率分别为（31.83 ± 16.36）%和（22.00 ± 15.48）%。转录组测序共筛选出698个差异表达基因，其中329个基因上调，369个基因下调；KEGG富集分析显示差异表达基因涉及Wnt信号通路。WB法检测结果显示，与对照组相比，单抗2G1组SOX2、OCT4、N-cadherin、p-β-catenin和c-Myc表达下调，E-cadherin表达上调（均P < 0.001）。结论：细胞膜表面HSP90β在MG-63球形细胞中高表达，靶向HSP90β的单抗2G1可抑制MG-63球形细胞的成球、迁移、侵袭及体内成瘤能力，并影响其干细胞标志物、EMT相关标志物及Wnt/β-catenin信号通路蛋白的表达水平，提示HSP90β可能参与调控MG-63球形细胞的OSC样特征，可作为骨肉瘤靶向治疗的潜在研究靶点。

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.

2022年广东医科大学青年培育基金（2XK22029/2001）；武汉市医学科研项目（WX21D51）；2025年度广东医科大学临床+基础科技创新专项计划