肿瘤细胞治疗研究
英文原题：Deletion of FAM76B histidine-rich region enhances macrophage-mediated osteosarcoma inhibition via liquid-liquid phase separation.
Deletion of FAM76B histidine-rich region enhances macrophage-mediated osteosarcoma inhibition via liquid-liquid phase separation.
我们的发现表明，靶向FAM76B相分离可能提供一种新的治疗策略，以对抗肿瘤诱导的免疫抑制，并增强T细胞免疫疗法在骨肉瘤及其他潜在实体瘤中的疗效。
背景：骨肉瘤（OS）是主要发生于年轻人群中的常见恶性肿瘤，常通过免疫抑制性髓系细胞介导的微环境逃避免疫抑制性T细胞疗法。本研究探讨FAM76B在OS中的作用及其对巨噬细胞极化和肿瘤免疫反应的影响。 方法：研究预测了FAM76B的内在无序区（IDR），并通过荧光共聚焦显微镜和荧光漂白后恢复（FRAP）实验考察其体外相分离特性，同时开展免疫荧光染色。利用CRISPR-Cas9和定点突变构建工程化骨肉瘤细胞，以研究FAM76B相分离对细胞功能的影响。通过克隆形成、MTT、划痕愈合实验、评估巨噬细胞功能和极化以及T细胞和NK细胞活性的流式细胞分析、皮下肿瘤异种移植模型和免疫组化等方法评估FAM76B作用。 结果：敲除FAM76B使巨噬细胞重编程，增强了OS中的抗肿瘤免疫。FAM76B缺失逆转了免疫抑制，增加T细胞浸润和活化。研究发现FAM76B呈现依赖富含组氨酸区（HRR）的相分离；删除该区域增强其诱导M1样巨噬细胞的能力，从而抑制OS细胞生长。 结论：研究结果提示，靶向FAM76B相分离可能成为一种新的治疗方法，以对抗肿瘤诱导的免疫抑制，并提高骨肉瘤及其他潜在实体瘤中T细胞疗法的疗效。
BACKGROUND: Osteosarcoma (OS), a prevalent malignancy primarily in young individuals, often evades T cell based immunotherapy due to an immunosuppressive myeloid cell-mediated microenvironment. This study investigated the role of FAM76B in OS and its impact on macrophage polarization and tumor immune response. METHODS: We predicted intrinsically disordered regions (IDRs) of FAM76B and explored its phase separation properties in vitro using fluorescence confocal microscopy and fluorescence recovery after photobleaching (FRAP) experiments. Immunofluorescence staining was also performed. We engineered osteosarcoma cells using CRISPR Cas9 and site directed mutagenesis to study the impact of FAM76B phase separation on cellular function. The effects of FAM76B were assessed through colony formation, MTT assays, wound healing assays, flow cytometry for macrophage function and polarization, T cell and NK cell activity, subcutaneous tumor xenograft models, and immunohistochemistry. RESULTS: Deletion of FAM76B reprogrammed macrophages, enhancing antitumor immunity in OS. Absence of FAM76B reversed immunosuppression, leading to increased T cell infiltration and activation. We found FAM76B exhibited histidine rich region (HRR) dependent phase separation; deletion of this region enhanced its ability to induce M1 like macrophages, inhibiting OS cell growth. CONCLUSION: Our findings suggest that targeting FAM76B phase separation may offer a novel therapeutic approach to counteract tumor induced immunosuppression and enhance efficacy of T cell based immunotherapy in osteosarcoma and potentially other solid tumors.
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