Table_1_Single-Cell Atlas Reveals Complexity of the Immunosuppressive Microenvironment of Initial and Recurrent Glioblastoma.docx

The Glioblastoma (GBM) immune microenvironment plays a critical role in tumor development, progression, and prognosis. A comprehensive understanding of the intricate milieu and its interactions remains unclear, and single-cell analysis is crucially needed. Leveraging mass cytometry (CyTOF), we analyzed immunocytes from 13 initial and three recurrent GBM samples and their matched peripheral blood mononuclear cells (pPBMCs). Using a panel of 30 markers, we provide a high-dimensional view of the complex GBM immune microenvironment. Hematoxylin and eosin staining and polychromatic immunofluorescence were used for verification of the key findings. In the initial and recurrent GBMs, glioma-associated microglia/macrophages (GAMs) constituted 59.05 and 27.87% of the immunocytes, respectively; programmed cell death-ligand 1 (PD-L1), T cell immunoglobulin domain and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), interleukin-10 (IL-10) and transforming growth factor-β (TGFβ) demonstrated different expression levels in the GAMs among the patients. GAMs could be subdivided into different subgroups with different phenotypes. Both the exhausted T cell and regulatory T (Treg) cell percentages were significantly higher in tumors than in pPBMCs. The natural killer (NK) cells that infiltrated into the tumor lesions expressed higher levels of CXC chemokine receptor 3 (CXCR3), as these cells expressed lower levels of interferon-γ (IFNγ). The immune microenvironment in the initial and recurrent GBMs displayed similar suppressive changes. Our study confirmed that GAMs, as the dominant infiltrating immunocytes, present great inter- and intra-tumoral heterogeneity and that GAMs, increased exhausted T cells, infiltrating Tregs, and nonfunctional NK cells contribute to local immune suppressive characteristics. Recurrent GBMs share similar immune signatures with the initial GBMs except the proportion of GAMs decreases.

胶质母细胞瘤（Glioblastoma, GBM）免疫微环境在肿瘤发生、进展及预后中发挥关键作用。目前对其复杂微环境及其相互作用的全面认知仍存在空白，亟需开展单细胞分析。本研究借助质谱流式细胞术（mass cytometry, CyTOF），对13例初诊胶质母细胞瘤、3例复发胶质母细胞瘤样本及其匹配的外周血单个核细胞（peripheral blood mononuclear cells, pPBMCs）中的免疫细胞进行了分析。通过包含30个标志物的检测panel，我们获得了胶质母细胞瘤免疫微环境的高维度全景视图。研究采用苏木精-伊红染色与多色免疫荧光技术对关键研究发现进行了验证。

在初诊与复发胶质母细胞瘤中，胶质瘤相关小胶质细胞/巨噬细胞（glioma-associated microglia/macrophages, GAMs）分别占免疫细胞总数的59.05%与27.87%；程序性死亡配体1（programmed cell death-ligand 1, PD-L1）、T细胞免疫球蛋白域和黏蛋白域-3（T cell immunoglobulin domain and mucin domain-3, TIM-3）、淋巴细胞活化基因-3（lymphocyte activation gene-3, LAG-3）、白细胞介素-10（interleukin-10, IL-10）及转化生长因子-β（transforming growth factor-β, TGFβ）在不同患者的GAMs中呈现差异化表达。GAMs可进一步划分为具有不同表型的多个亚群。肿瘤组织内耗竭性T细胞与调节性T（regulatory T, Treg）细胞的占比均显著高于匹配的外周血单个核细胞。浸润至肿瘤病灶的自然杀伤（natural killer, NK）细胞高表达CXC趋化因子受体3（CXC chemokine receptor 3, CXCR3），而此类细胞的干扰素-γ（interferon-γ, IFNγ）表达水平较低。初诊与复发胶质母细胞瘤的免疫微环境均呈现相似的免疫抑制特征变化。

本研究证实，作为主要浸润性免疫细胞群的GAMs具有显著的瘤间及瘤内异质性；GAMs、增多的耗竭性T细胞、浸润性调节性T细胞以及功能失调的NK细胞共同促成了局部的免疫抑制特性。除GAMs占比下降外，复发胶质母细胞瘤与初诊胶质母细胞瘤具有相似的免疫特征。