低氧微环境通过 FCGR2B 介导的巨噬细胞极化促进胶质母瘤放疗耐受的机制研究

Hypoxic Microenvironment Drives FCGR2B-Mediated Macrophage Polarization to Promote Radiotherapy Resistance in Glioblastoma

摘要:

目的 探讨低氧微环境是否通过调控FCGR2B影响肿瘤相关巨噬细胞(TAMs)极化,进而介导胶质母细胞瘤(Glioblastoma multiforme,GBM)的放疗抵抗,并评估靶向FCGR2B的增敏潜力。方法 基于CGGA数据库中 GBM转录组数据,采用一致性聚类分析低氧相关基因表达模式,并进行生存分析。利用GEPIA2、TIMER2.0及单细胞RNA测序数据库分析FCGR2B的表达特征和免疫相关性。体外实验中,将THP-1细胞经PMA诱导分化为巨噬细胞,通过shRNA慢病毒敲低FCGR2B表达,在常氧(21% O2)或低氧(2% O2)条件下培养,使用流式细胞术检测细胞表型(CD163⁺比例)和FCGR2B表达,qPCR和Western blot检测M1/M2标志物(CD86, NOS2, CD163, ARG1)及FCGR2B的蛋白水平。动物实验中,构建C57BL/6小鼠原位GL261胶质瘤模型,设立对照组、放疗组、FCGR2B单抗组及放疗联合FCGR2B单抗组,比较肿瘤体积、生存期及免疫细胞浸润情况。体内实验构建C57BL/6小鼠原位GL261-luc GBM模型,设置 WT 组、WT+RT组、FCGR2B基因敲除+RT组(fcgr2b^-/-+RT)、isotype+RT 组,FCGR2B mAb +RT组,通过小动物活体成像、H E染色评估肿瘤体积,流式细胞术分析肿瘤浸润F4/80+^CD206+细胞。结果 一致性聚类将GBM分为低氧特征高表达的C1簇和低表达的C2簇,C1簇患者预后显著差于C2簇(P<0.0001)。FCGR2B在C1簇中显著高表达,且其高表达与患者不良预后相关(P<0.05)。单细胞测序分析证实FCGR2B特异性表达于TAMs,尤其在M2型TAMs中富集,并与CD163、ARG1等M2标志物表达呈强正相关(均P<0.05)。体外实验表明,低氧条件可显著上调巨噬细胞FCGR2B的表达(P<0.001),并促进其向M2型极化(CD163⁺细胞比例及CD163、ARG1 mRNA表达均显著升高,P<0.05)。而敲低FCGR2B后,低氧诱导的M2极化效应被显著抑制(CD163⁺细胞比例及CD163、ARG1 mRNA表达均显著下降,P<0.01)。体内实验证明,与WT+RT组相比,fcgr2b-/-^+RT组小鼠的肿瘤生长均受到显著抑制(P<0.01),肿瘤内F4/80+^CD206+细胞浸润比例显著降低(P<0.01)。FCGR2B mAb +RT组小鼠肿瘤生长及肿瘤内F4/80+^CD206+细胞浸润比例显著低于isotype+RT 组 。结论 低氧微环境通过上调FCGR2B表达驱动TAMs向M2型极化,从而促进GBM的放疗抵抗。靶向FCGR2B能有效重塑免疫微环境,增强放疗敏感性,为GBM的治疗提供了新的潜在靶点。

Abstract:

Objective To investigate whether the hypoxic microenvironment regulates FCGR2B to influence tumor-associated macrophage (TAM) polarization, thereby mediating radiotherapy resistance in glioblastoma multiforme (GBM), and to evaluate the radiosensitizing potential of targeting FCGR2B. Methods Transcriptomic data of GBM from the CGGA database were analyzed using consensus clustering to identify hypoxia-associated gene expression patterns, followed by survival analysis. FCGR2B expression features and immune correlations were assessed using GEPIA2, TIMER2.0, and single-cell RNA sequencing datasets. In vitro, THP-1 cells were differentiated into macrophages with PMA, and FCGR2B was silenced via shRNA lentiviral transduction. Cells were cultured under 21% O? or 2% O?, followed by flow cytometry to assess phenotype (CD163? proportion) and FCGR2B expression, and qPCR and Western blotting to measure M1/M2 markers (CD86, NOS2, CD163, ARG1) and FCGR2B protein levels. In vivo, orthotopic GL261 GBM models were established in C57BL/6 mice, including wild-type (WT), WT+RT, FCGR2B knockout + RT (fcgr2b^?/? + RT), isotype control + RT (isotype + RT), and FCGR2B mAb + RT groups. Tumor volume was assessed by small animal in vivo imaging and hematoxylin-eosin (H E) staining. Tumor-infiltrating F4/80?CD206? cells were analyzed by flow cytometry. Results Consensus clustering divided GBM into a hypoxia-high (C1) and a hypoxia-low (C2) cluster, with C1 patients showing significantly poorer survival than C2 (P<0.0001). FCGR2B expression was elevated in C1 and associated with worse prognosis (P<0.05). Single-cell analyses confirmed that FCGR2B was specifically enriched in TAMs, particularly M2-like TAMs, and strongly correlated with M2 markers CD163 and ARG1 (both P<0.05). In vitro, hypoxia significantly upregulated FCGR2B expression (P<0.001) and promoted M2 polarization (increased CD163? proportion and CD163、ARG1 expression, P<0.05). FCGR2B knockdown abrogated hypoxia-induced M2 polarization (reduced CD163? proportion and CD163、ARG1 expression, P<0.01). In vivo, compared with WT+RT, the fcgr2b^?/?+RT group showed markedly inhibited tumor growth (P<0.01) and reduced F4/80?CD206? infiltration (P<0.01). Similarly, FCGR2B mAb +RT significantly suppressed tumor progression and TAM infiltration compared with isotype+RT. Conclusion The hypoxic microenvironment drives TAM M2 polarization via upregulation of FCGR2B, thereby promoting GBM radiotherapy resistance. Targeting FCGR2B effectively reprograms the immune microenvironment and enhances radiosensitivity, highlighting FCGR2B as a promising therapeutic target for GBM.