DataSheet_1_Functional characterization of TSPAN7 as a novel indicator for immunotherapy in glioma.csv

Glioma is the most common primary malignant tumor of the central nervous system in clinical practice. Most adult diffuse gliomas have poor efficacy after standard treatment, especially glioblastoma. With the in-depth understanding of brain immune microenvironment, immunotherapy as a new treatment has attracted much attention. In this study, through analyzing a large number of glioma cohorts, we reported that TSPAN7, a member of the tetraspanin family, decreased in high-grade gliomas and low expression was associated with poor prognosis in glioma patients. Meanwhile, the expression pattern of TSPAN7 was verified in glioma clinical samples and glioma cell lines by qPCR, Western Blotting and immunofluorescence. In addition, functional enrichment analysis showed that cell proliferation, EMT, angiogenesis, DNA repair and MAPK signaling pathways were activated in the TSPAN7 lower expression subgroup. Lentiviral plasmids were used to overexpress TSPAN7 in U87 and LN229 glioma cell lines to explore the anti-tumor role of TSPAN7 in glioma. Moreover, by analyzing the relationship between TSPAN7 expression and immune cell infiltration in multiple datasets, we found that TSPAN7 was significantly negatively correlated with the immune infiltration of tumor-related macrophages, especially M2-type macrophages. Further analysis of immune checkpoints showed that, the expression level of TSPAN7 was negatively correlated with the expression of PD-1, PD-L1 and CTLA-4. Using an independent anti-PD-1 immunotherapy cohorts of GBM, we demonstrated that TSPAN7 expression may had a synergistic effect with PD-L1 on the response to immunotherapy. Based on the above findings, we speculate that TSPAN7 can serve as a biomarker for prognosis and a potential immunotherapy target in glioma patients.

胶质瘤是临床中最常见的中枢神经系统原发性恶性肿瘤。多数成人弥漫性胶质瘤在接受标准治疗后疗效不佳，其中胶质母细胞瘤（GBM）的预后尤为堪忧。随着对脑部免疫微环境研究的不断深入，免疫治疗作为新型治疗手段受到学界广泛关注。本研究通过分析大量胶质瘤队列数据，发现四跨膜蛋白（tetraspanin）家族成员TSPAN7在高级别胶质瘤中表达下调，且TSPAN7低表达与胶质瘤患者的不良预后显著相关。同时，本研究通过实时荧光定量PCR（qPCR）、蛋白质印迹（Western Blotting）及免疫荧光实验，在胶质瘤临床样本与胶质瘤细胞系中验证了TSPAN7的表达模式。此外，功能富集分析结果显示，在TSPAN7低表达亚组中，细胞增殖、上皮间质转化（EMT）、血管生成、DNA损伤修复及丝裂原活化蛋白激酶（MAPK）信号通路均被激活。本研究构建慢病毒质粒，在U87与LN229胶质瘤细胞系中过表达TSPAN7，以探究其在胶质瘤中的抗肿瘤作用。进一步通过分析多组数据集内TSPAN7表达与免疫细胞浸润的关联，本研究发现TSPAN7的表达水平与肿瘤相关巨噬细胞的免疫浸润程度呈显著负相关，尤以M2型巨噬细胞为甚。针对免疫检查点的进一步分析显示，TSPAN7的表达水平与PD-1、PD-L1及CTLA-4的表达均呈负相关。本研究利用独立的胶质母细胞瘤抗PD-1免疫治疗队列数据，证实TSPAN7的表达与PD-L1可能在免疫治疗响应中发挥协同作用。基于上述研究结果，本研究推测TSPAN7可作为胶质瘤患者预后评估的生物标志物及潜在的免疫治疗靶点。