Table 1_Immune intrinsic escape signature stratifies prognosis, characterizes the tumor immune microenvironment, and identifies tumorigenic PPP1R8 in glioblastoma multiforme patients.docx

BackgroundGlioblastoma (GBM) is a highly aggressive brain tumor with poor prognosis and limited response to immunotherapy. Immune escape-related genes (IERGs) are increasingly recognized as critical regulators of tumor progression and immune evasion. However, their prognostic value in GBM remains unclear. This study aims to evaluate the clinical relevance of IERGs and develop a predictive gene signature to guide prognosis and characterize the tumor immune microenvironment (TIME).
MethodsWe performed a comprehensive analysis of IERGs using the TCGA GBM dataset. Prognostic IERGs were identified through univariate Cox regression, and a multivariate Cox model was used to develop a prognostic signature. Risk scores (IEScore) were calculated to classify patients into high- and low-risk groups. The signature was validated in two independent GBM cohorts. Its prognostic independence was assessed after adjusting for clinicopathological features. Receiver operating characteristic (ROC) analysis confirmed the signature’s reliability. TIME analysis was carried out using multiple deconvolution algorithms. Additionally, functional assays including CCK8, cell cycle, and apoptosis assays were conducted on PPP1R8-silenced U251 cells using CRISPR/Cas9 technology
ResultsThirty-six IERGs were associated with GBM outcomes, with 20 linked to poor survival and 16 to better outcomes. Key genes, including STAT2, IFNGR2, and PPP1R8, formed a robust prognostic signature. High-risk patients had significantly poorer overall survival (OS) compared to low-risk patients. The signature showed strong predictive power with AUC values of 0.68, 0.73, and 0.76 for 2-, 3-, and 5-year survival, respectively. Validation in two independent cohorts confirmed its robustness. Immune cell infiltration analysis revealed distinct patterns in high- and low-risk groups, with the high-risk group showing a more aggressive and immunosuppressive tumor microenvironment. The signature also effectively stratified low-grade glioma patients across four independent datasets. Knockout of PPP1R8 in GBM cells using CRISPR/Cas9 inhibited cell proliferation and increased apoptosis.
ConclusionThe IERGs-based signature offers reliable prognostication for GBM, validated across multiple datasets. It can guide patient stratification and inform therapeutic decisions for GBM and potentially low-grade gliomas (LGG). Furthermore, we identify PPP1R8 as a key regulator of GBM cell proliferation and growth, providing insights into the immune microenvironment’s role in GBM progression.

背景 胶质母细胞瘤（Glioblastoma, GBM）是一种恶性程度极高的脑肿瘤，预后极差且对免疫治疗响应有限。免疫逃逸相关基因（Immune escape-related genes, IERGs）作为肿瘤进展与免疫逃逸的关键调控因子，正受到越来越多的关注。然而，其在胶质母细胞瘤中的预后价值仍不明确。本研究旨在评估免疫逃逸相关基因的临床相关性，开发一款预测性基因标签以指导预后评估，并解析肿瘤免疫微环境（tumor immune microenvironment, TIME）。
方法 本研究利用癌症基因组图谱（The Cancer Genome Atlas, TCGA）胶质母细胞瘤数据集对免疫逃逸相关基因进行了全面分析。通过单因素Cox回归筛选预后相关免疫逃逸相关基因，并采用多因素Cox模型构建预后标签。计算风险评分（IEScore）以将患者划分为高风险组与低风险组。该标签在两个独立的胶质母细胞瘤队列中得到验证。在校正临床病理特征后，评估了该标签的预后独立性。受试者工作特征（Receiver Operating Characteristic, ROC）分析证实了该标签的可靠性。采用多种反卷积算法开展肿瘤免疫微环境分析。此外，本研究借助成簇规律间隔短回文重复序列/相关蛋白9（CRISPR/Cas9）技术，对沉默PPP1R8基因的U251细胞进行了包括细胞计数试剂盒-8（Cell Counting Kit-8, CCK8）、细胞周期实验及细胞凋亡实验在内的功能学验证。
结果 共有36个免疫逃逸相关基因与胶质母细胞瘤预后相关，其中20个基因与不良生存相关，16个基因与更佳预后相关。包括STAT2、IFNGR2及PPP1R8在内的关键基因构建了一款稳定性良好的预后标签。高风险组患者的总生存期（overall survival, OS）显著低于低风险组。该标签具有较强的预测能力，2年、3年及5年生存率对应的曲线下面积（Area Under Curve, AUC）分别为0.68、0.73及0.76。在两个独立队列中的验证结果证实了该标签的稳定性。免疫细胞浸润分析显示，高、低风险组具有截然不同的免疫浸润模式，高风险组呈现出更具侵袭性且免疫抑制性更强的肿瘤微环境。该标签还可在四个独立数据集的低级别胶质瘤患者中实现有效分层。利用CRISPR/Cas9技术敲除胶质母细胞瘤细胞中的PPP1R8基因，可抑制细胞增殖并促进细胞凋亡。
结论 基于免疫逃逸相关基因构建的预后标签可为胶质母细胞瘤提供可靠的预后评估，该标签已在多组数据集内得到验证。其可指导胶质母细胞瘤乃至潜在低级别胶质瘤（low-grade gliomas, LGG）患者的分层治疗，并为治疗决策提供参考。此外，本研究证实PPP1R8是胶质母细胞瘤细胞增殖与生长的关键调控因子，为解析肿瘤免疫微环境在胶质母细胞瘤进展中的作用提供了新的见解。