背景:Anoikis是由细胞与细胞外基质(ECM)的粘附丧失引起的程序性细胞死亡的一种特殊形式。抗肛门凋亡的获得是癌细胞侵袭的重要标志,转移,治疗抵抗,和复发。尽管目前的研究已经确定了多种调节抗肛门凋亡的因素,胶质母细胞瘤(GBM)中失巢凋亡介导的肿瘤微环境(TME)的病理机制仍未被研究.
方法:利用单细胞RNA测序(scRNA-seq)数据并采用非负矩阵分解(NMF),我们鉴定并表征了具有明显失巢凋亡相关基因特征的TME细胞簇.使用TCGA和CGGA数据集进行预后和治疗反应分析,以评估不同TME细胞簇的临床意义。从空间转录组RNA测序(stRNA-seq)数据推断BRMS1+小胶质细胞与肿瘤细胞之间的空间关系。模拟肿瘤免疫微环境,用小胶质细胞(HMC3)和GBM细胞(U118/U251)进行共培养实验,用BRMS1过表达慢病毒转染小胶质细胞。Westernblot或ELISA检测BRMS1、M2巨噬细胞特异性标志物,PI3K/AKT信号蛋白,和凋亡相关蛋白。用CCK-8、集落形成、和细胞凋亡试验,而肿瘤细胞的侵袭和迁移能力使用Transwell测定法进行评估。
结果:基于NMF的分析成功鉴定出具有不同基因特征的CD8+T细胞和小胶质细胞簇。轨迹分析,细胞通讯,和基因调控网络分析共同表明,失巢凋亡介导的TME细胞簇可以通过各种机制影响肿瘤细胞的发育。值得注意的是,BRMS1+AP-Mic表现出M2巨噬细胞表型,并与恶性细胞有显著的细胞通讯。此外,BRMS1+AP-Mic在TCGA和CGGA数据集中的高表达与较差的生存结局相关,表明其对免疫疗法的有害影响。BRMS1在小胶质细胞中的上调可能导致M2巨噬细胞极化,通过SPP1/CD44介导的细胞相互作用激活PI3K/AKT信号通路,抑制肿瘤细胞凋亡,促进肿瘤的增殖和侵袭。
结论:这项开创性的研究使用基于NMF的分析来揭示GBM中失巢凋亡调节的TME对预后和免疫治疗反应的重要预测价值。BRMS1+小胶质细胞为深入了解GBM的免疫抑制微环境提供了新的视角,并可能成为未来潜在的治疗靶点。
BACKGROUND: Anoikis is a specialized form of programmed cell death induced by the loss of cell adhesion to the extracellular matrix (ECM). Acquisition of anoikis resistance is a significant marker for cancer cell invasion, metastasis, therapy resistance, and recurrence. Although current research has identified multiple factors that regulate anoikis resistance, the pathological mechanisms of anoikis-mediated tumor microenvironment (TME) in glioblastoma (GBM) remain largely unexplored.
METHODS: Utilizing single-cell RNA sequencing (scRNA-seq) data and employing non-negative matrix factorization (
NMF), we identified and characterized TME cell clusters with distinct anoikis-associated gene signatures. Prognostic and therapeutic response analyses were conducted using TCGA and CGGA datasets to assess the clinical significance of different TME cell clusters. The spatial relationship between BRMS1 + microglia and tumor cells was inferred from spatial transcriptome RNA sequencing (stRNA-seq) data. To simulate the tumor immune microenvironment, co-culture experiments were performed with microglia (HMC3) and GBM cells (U118/U251), and microglia were transfected with a BRMS1 overexpression lentivirus. Western blot or ELISA were used to detect BRMS1, M2 macrophage-specific markers, PI3K/AKT signaling proteins, and apoptosis-related proteins. The proliferation and apoptosis capabilities of tumor cells were evaluated using CCK-8, colony formation, and apoptosis assays, while the invasive and migratory abilities of tumor cells were assessed using Transwell assays.
RESULTS: NMF-based analysis successfully identified CD8 + T cell and microglia cell clusters with distinct gene signature characteristics. Trajectory analysis, cell communication, and gene regulatory network analyses collectively indicated that anoikis-mediated TME cell clusters can influence tumor cell development through various mechanisms. Notably, BRMS1 + AP-Mic exhibited an M2 macrophage phenotype and had significant cell communication with malignant cells. Moreover, high expression of BRMS1 + AP-Mic in TCGA and CGGA datasets was associated with poorer survival outcomes, indicating its detrimental impact on immunotherapy. Upregulation of BRMS1 in microglia may lead to M2 macrophage polarization, activate the PI3K/AKT signaling pathway through SPP1/CD44-mediated cell interactions, inhibit tumor cell apoptosis, and promote tumor proliferation and invasion.
CONCLUSIONS: This pioneering study used
NMF-based analysis to reveal the important predictive value of anoikis-regulated TME in GBM for prognosis and immunotherapeutic response. BRMS1 + microglial cells provide a new perspective for a deeper understanding of the immunosuppressive microenvironment of GBM and could serve as a potential therapeutic target in the future.