Functional characterization of TSPAN7 as a novel indicator for immunotherapy in glioma

Abstract

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.

Keywords: glioma; immune infiltration; immunotherapy; malignant progression; single-cell RNA sequencing.

Figure 1

Clinical and molecular characteristics of TSPAN7 in gliomas. (A) The expression of TSPAN7 in multiple cancer from TCGA dataset. LAML, acute myeloid leukemia; UVM, Uveal Melanoma; KIRP, kidney renal papillary cell carcinoma; BLCA, bladder urothelial carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; TGCT, testicular germ cell tumors; LIHC, liver hepatocellular carcinoma; BRCA, breast invasive carcinoma; UCEC, uterine corpus endometrial carcinoma; THYM, thymoma; CHOL, cholangiocarcinoma; MESO, Mesothelioma; SARC, sarcoma; STAD, stomach adenocarcinoma; COAD, colon adenocarcinoma; PRAD, prostate adenocarcinoma; LUAD, lung adenocarcinoma; HNSC, head and neck squamous cell carcinoma; ESCA, esophageal carcinoma; READ, rectum adenocarcinoma; PAAD, pancreatic adenocarcinoma; OV, ovarian serous cystadenocarcinoma; SKCM, skin cutaneous melanoma; THCA, thyroid carcinoma; LUSC, lung squamous cell carcinoma; UCS, uterine carcinosarcoma; KIRC, kidney renal clear cell carcinoma; ACC, adrenocortical carcinoma; KICH, kidney chromophobe; PCPG, pheochromocytoma and paraganglioma; GBM, glioblastoma multiforme; LGG, brain lower grade glioma. B, CThe expression of TSPAN7 stratified by WHO pathological grades and types. (D, E) The expression of TSPAN7 in 1p19q codeletion and non-codeletion glioma and IDH mutant and wild-type status. (F) The expression of TSPAN7 in IDH mutation status combined with different pathological grades. (G) The expression of TSPAN7 stratified by MGMT promoter methylation status. (H) The mRNA relative level of TSPAN7 in clinical glioma samples according to different WHO pathological grades. (I, J) Representative images of Western Blot for TSPAN7 in normal brain tissue and different pathological grades. (K) Representative images of IF staining for TSPAN7 in normal brain tissue and different WHO pathological grades of glioma. *p<.05, **p <.01, ***p <.001, ****p <.0001, ns: no statistics.

Figure 2

The expression of TSPAN7 in different glioma subtypes (A-C) TSPAN7 expression level in different molecular subtypes in CGGA_325, CGGA_691 and TCGA datasets based on bulk expression. (D-E) The cells were categorized into seven clusters and scatter plots represent TSPAN7 expression distribution in seven clusters. (F) The expression of TSPAN7 in seven clusters. (G) single cell was divided into Scissor+ cell or Scissor- cell 3 groups according to the prognosis by scissor algorithm. (H) The expression of TSPAN7 in Scissor+ cell and Scissor- cell. (I) Tumor cells were extracted and categorized into four different cell clusters based on scRNA expression level. ****p <.0001.

Figure 3

The relationship between TSPAN7 expression and survival for glioma. (A-F) Kaplan–Meier plots of TSPAN7 in (A) GSE16011, (B) GSE108474, (C) GSE43289, (D) TCGA, (E) CGGA_325 and (F) CGGA_693datasets. (G-I) The forest diagrams represent the multi-factor COX regression analysis, in which the variables include TSPAN7, gender, age, pathological grade, IDH mutation status and MGMT methylation status in TCGA (G), CGGA_325 (H), CGGA_693 (I).

Figure 4

Functional enrichment analysis between the TSPAN7 high- and low‐expression groups in gliomas. (A) Differential expression genes (DEGs). (B) GO analysis of the up-regulated gene in the high TSPAN7 subgroup and the down-regulated gene in the high TSPAN7 subgroup. (C) The relationship between TSPAN7 and proliferation, migration, DNA repair and MAPK signal pathway in the TCGA datasets. (D) Differences in enrichment scores between TSPAN7 high and low expression subgroups in cell proliferation, EMT, angiogenesis, hypoxia, DNA repair and MAPK signaling pathways. ****p <.0001.

Figure 5

TSPAN7 expression in GBM is protective against cell proliferation, viability and migration in vitro. (A) The expression level of TSPAN7 in the three glioma cell lines and normal glia cells were detected by Western blot. (B, C) The mRNA expression levels of TSPAN7 in normal, control and TSPAN7-overexpressing cell plasmid transfection were detected by qPCR and Western blot. (D, E) CCK-8 assays showed the difference of proliferation capacity between wildtype, LV-Ctrl and TSPAN7-overexpression in U87 and LN229 cell lines. (F) Cell cycle differences between TSPAN7-overexpression and control subgroup by flow cytometry in U87 and LN229 cell lines. (G) Wound-healing assays showed that cell migration ability between wildtype, LV-Ctrl and TSPAN7 overexpression in U87 cell line, *p <.05, ***p <.001, ns: no statistics.

Figure 6

The relationships between TSPAN7 expression and immune infiltration of 22 immune cells in glioma immune microenvironment. (A, B) Relative immune cell infiltration level of 22 immune cells between TSPAN7 high expression and low expression subgroup in TCGA and CGGA datasets. (C) Correlation analysis of TSPAN7 with M1 type and M2 type macrophage related specific markers in TCGA dataset.

Figure 7

Low TSPAN7 patients are more practical to anti-PD-1 immunotherapy. (A) Correlation analysis of TSPAN7 expression with immune checkpoint markers in TCGA dataset. (B) Relative expression level differences of different immune checkpoints between TSPAN7 high expression and low expression subgroup in TCGA dataset. (C, D) Differences in the proportion of patient treatment response to anti-PD-1 immunotherapy and survival differences between TSPAN7 high and low expression subgroups. (E, F) Differences in the proportion of patient treatment response to anti-PD-1 immunotherapy and survival differences between TSPAN7 high or low expression subgroups combined with PDL1 high or low expression. *p <.05, **p <.01, ****p <.0001, ns, no statistics.