Graphical abstract graphic file with name fx1.jpg [41]Open in a new tab Highlights * • TSPAN4 is highly expressed in GBM and is notably correlated with poor prognosis * • TSPAN4 plays an oncogene role in GBM * • TSPAN4 interacts with EGFR and positively regulates its stability in GBM cells * • TSPAN4 promotes GBM progression by activating the EGFR signaling pathway __________________________________________________________________ Biological sciences; Molecular biology; Molecular medicine; Natural sciences Introduction Glioblastoma (GBM), the most lethal among primary brain tumors, is characterized by its highly malignant and aggressive nature. The prognosis is grim, with a majority of patients succumbing within a year, and only a mere 5% managing to extend their survival beyond 5 years[42]^1^,[43]^2 Currently, the principal treatment for GBM is still surgical resection, combined with radiotherapy and chemotherapy.[44]^3^,[45]^4^,[46]^5 Due to the aggressive growth characteristics, most GBMs are refractory to complete resection on a wide scale, and the residual tumor cells become the root cause of recurrence, which leads to a poor prognosis for some GBM patients.[47]^6 Therefore, a more in-depth understanding of the mechanisms of GBM genesis facilitates the development of new therapeutic strategies to alleviate the progression of GBM. TSPAN4, a member of the TSPAN family, is required for the formation of migrasomes, a recently identified vesicular organelle linked to cell migration.[48]^7^,[49]^8 Activation of the TGF-β1 signaling pathway induces TSPAN4 expression and migrasome production, facilitating the progression of proliferative vitreoretinopathy.[50]^9 Moreover, TSPAN4 overexpression significantly confers resistance to paclitaxel and cisplatin in esophageal squamous cell carcinoma (ESCC).[51]^10 Elevated TSPAN4 levels are associated with poor prognosis in patients with gastric cancer and GBM.[52]^11^,[53]^12 Currently, research on the specific functions of TSPAN4, particularly in cancers, is limited. Given the adverse impact of TSPAN4 expression on GBM prognosis, investigating its potential oncogenic role in GBM is worthy of consideration. Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein belonging to the tyrosine kinase superfamily of receptors.[54]^13 It is arguably the most prominent signaling pathway, capable of activating multiple downstream pathways, including ERK and mTOR, thereby promoting GBM progression.[55]^14^,[56]^15 Targeting EGFR represents a promising therapeutic strategy. However, EGFR mutations, such as EGFRvIII, have been identified in GBM, rendering tyrosine kinase inhibitor (TKI) therapy ineffective.[57]^16 Therefore, further exploration of novel regulatory mechanisms of EGFR and the combination of EGFR inhibitors is warranted. Here, we have demonstrated that elevated expression of TSPAN4 correlates with adverse prognosis and treatment outcomes in glioma patients. TSPAN4 knockdown suppressed GBM progression in vivo and in vitro, leading to cell-cycle arrest at the G2/M phase by downregulating the expression of CDK1, CDK2, and CCNB1. Furthermore, our findings unveiled that TSPAN4 facilitated GBM progression via activation of the EGFR signaling pathway, suggesting that targeting TSPAN4 could offer significant clinical benefits for patients. Results Elevated expression of TSPAN4 is associated with progression and poor prognosis in gliomas To investigate the essential role of TSPAN4 in cancer, we conducted pan-cancer and univariate Cox regression analyses from the TCGA and GTEx databases to examine the mRNA expression of TSPAN4 across various cancer types and its association with prognosis. Remarkably, we observed a significant upregulation of TSPAN4 mRNA expression in gliomas, particularly in GBM, which posed a significant risk to the overall survival (OS) of glioma patients ([58]Figures 1A, [59]S1A, and S1B). Furthermore, our findings were corroborated by analyses conducted on the CGGA and [60]GSE16011 cohorts. ([61]Figures 1B and 1C). To assess the association between TSPAN4 expression and survival across different glioma subtypes, K-M curves demonstrated that patients with high TSPAN4 expression had a poorer prognosis for overall survival, whether diagnosed with LGG or GBM ([62]Figures 1D and 1E). Notably, the hazard ratio (HR) was higher in GBM (HR = 2.119, p < 0.001) compared to LGG (HR = 1.344, p = 0.004) ([63]Figure S1B). Time-dependent receiver operating characteristic (ROC) curves illustrated that TSPAN4 expression accurately predicted 1-, 3-, and 5-year overall survival rates with AUC values of 0.822, 0.827, and 0.733, respectively ([64]Figure 1F). Furthermore, we conducted TSPAN4 immunohistochemistry (IHC) staining on GBM tissue microarrays, comprising 60 GBM and 8 normal brain tissue biopsies, to examine TSPAN4 expression levels in GBM. Consistent with the previous findings, we noted increased TSPAN4 expression in GBM compared to normal brain tissue ([65]Figures 1G and 1H). These data suggest that TSPAN4 is highly expressed and correlated with tumor progression and poor prognosis in glioma patients. Figure 1. [66]Figure 1 [67]Open in a new tab TSPAN4 is highly expressed in gliomas and is associated with poor prognosis (A–C) TSPAN4 mRNA expression in normal, LGG, and GBM tissues from TCGA, CGGA, and [68]GSE16011 databases, respectively. (D and E) Kaplan-Meier curves show the relationship between TSPAN4 expression and overall survival in LGG and GBM patients from the TCGA databases. (F) Time-dependent ROC curves showing the ability of TSPAN4 expression to predict 1-, 3-, and 5-year overall survival in glioma patients from the TCGA database. (G) Representative immunohistochemistry staining of TSPAN4 expression on a GBM tissue microarray. Scale bar, 50 μm. (H) Analysis of IHC score of TSPAN4 expression in normal brain and GBM from the GBM tissue microarray. (I) Univariate Cox regression analyses of risk factors affecting the survival of glioma patients. (J–N) The correlation between TSPAN4 mRNA expression level and age (J), WHO grade (K), histological type (L), IDH status (M), and primary therapy outcome (N) in glioma patients. Data in H are presented as the mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Two-tailed Student’s t test. To further explicate the clinical implication of TSPAN4, we evaluated the relationship between TSPAN4 expression and clinical-pathological parameters in glioma patients. First, we executed a univariate Cox regression analysis using the clinical data from the TCGA database. It turned out that age, WHO grade, histological type, IDH status, and therapy outcome were independent risk factors that threatened the survival of glioma patients ([69]Figure 1I). Strikingly, enlarged TSPAN4 expression was significantly associated with age, WHO grade, histological type, IDH status, and therapy outcome of glioma patients ([70]Figures 1J–1N), whereas there was no significant relationship with 1p/19q codeletion, gender, and race ([71]Figures S1C–S1E). These findings indicate that TSPAN4 is implicated in the grade, histologic subtype, and treatment outcome of glioma patients. To further investigate the association between TSPAN4 expression and treatment efficacy in glioma patients, we utilized progression-free interval (PFI) and disease-specific survival (DSS) data from the TCGA database, which can partially reflect treatment effectiveness in this patient population. Our findings revealed that glioma patients with high TSPAN4 expression exhibited significantly shorter PFI and DSS compared to those with low expression, both in LGG and GBM ([72]Figures S1F–S1I). Additionally, K-M survival analysis of 195 glioma patients treated with temozolomide (TMZ) or radiotherapy also showed shorter progression-free survival (PFS) in patients with high TSPAN4 expression ([73]Figure S1J). These results suggest that high TSPAN4 expression is associated with poorer treatment outcomes, including chemotherapy and radiotherapy. TSPAN4 facilitates tumorigenic potentials of GBM cells in vitro To examine whether TSPAN4 exerted oncogenic roles in GBM, we constructed TSPAN4 knockdown stable cell lines with two independent shRNA individually to evaluate the effect of TSPAN4 on GBM cell proliferation. Quantitative polymerase chain reaction (qPCR) and western blot (WB) assays confirmed the knockdown efficiency of TSPAN4 in 3 GBM cells, including U87, U251, and primary human GBM cells (GBM1) ([74]Figures 2A and [75]S2A). CCK-8 cell proliferation and colony formation assays showed that TSPAN4 knockdown significantly suppressed GBM cell proliferation ([76]Figures 2B and 2C). Furthermore, the transwell invasion assay demonstrated that TSPAN4 knockdown in GBM cells prominently reduced the ability of GBM cell invasion ([77]Figure 2D). Subsequently, Ki67 and EdU staining assays revealed that TSPAN4 depletion severely decreased the positive rate of Ki67 and EdU in U87 and U251 compared to the control group, indicating that TSPAN4 knockdown limited the growth rate of GBM cells ([78]Figures 2E and [79]S2B). Additionally, we also observed changes in the cell cycle and cell apoptosis upon TSPAN4 depletion in GBM cells. Cell cycle and apoptosis assays revealed that TSPAN4 knockdown induced cell cycle G2/M arrest and promoted apoptosis, implying that TSPAN4 inhibited GBM cell proliferation by regulating cell cycle and apoptosis ([80]Figures 2F, 2G, and [81]S2C). Figure 2. [82]Figure 2 [83]Open in a new tab TSPAN4 knockdown inhibits GBM cell proliferation and invasion and induces cell-cycle arrest in vitro (A) WB confirms the knockdown efficiency of TSPAN4 at the protein levels in U87, U251, and GBM1 cells. (B and C) CCK-8 and colony formation assays show the effect of TSPAN4 knockdown on cell proliferation in U87, U251, and GBM1 cells. (D) Transwell assay shows the effect of TSPAN4 knockdown on invasion in U87, U251, and GBM1 cells. Scale bar, 50 μm. (E) Representative images of EdU staining and the comparison of EdU-positive ratio upon TSPAN4 knockdown in U87 and U251 cells. Scale bar, 100 μm. (F and G) Flow cytometry shows the effect of TSPAN4 knockdown on the cell cycle of U87 and U251. (H) WB validates the restored TSPAN4 expression upon TSPAN4 knockdown in U87 and U251 cells. (I) CCK-8 assay shows the effect of restored TSPAN4 expression on cell proliferation upon TSPAN4 knockdown in U87 and U251 cells. Data in (B–E) and I are presented as the mean ± SD (n = 3). ^∗∗∗p < 0.001. One-way ANOVA with Tukey’s test. To confirm the crucial role of TSPAN4 in GBM cells, we introduced the shRNA-resistant synonymous mutant of TSPAN4 into U87 and U251 cells following TSPAN4 knockdown and verified the restored TSPAN4 expression ([84]Figure 2H). CCK-8 cell proliferation assays revealed that reintroducing TSPAN4 restored GBM cell proliferation upon TSPAN4 knockdown ([85]Figure 2I). These results indicate that TSPAN4 knockdown dramatically inhibits the GBM cell proliferation. In addition to loss-of-function experiments, we transfected U87, U251, and GBM1 cells with vector and TSPAN4-FLAG plasmids and confirmed the efficiency of TSPAN4 overexpression by WB ([86]Figure 3A). Contrary to the results of TSPAN4 knockdown, overexpression of TSPAN4 enhanced GBM cell viability, colony formation, invasion, and the positive rates of EdU and Ki67, while suppressing apoptosis ([87]Figures 3B–3F and [88]S2D). Collectively, these findings indicate that TSPAN4 enhances the tumorigenic properties of GBM cells by promoting proliferation, invasion, cell cycle progression, and inhibiting apoptosis in vitro. Figure 3. [89]Figure 3 [90]Open in a new tab TSPAN4 overexpression promotes GBM cell proliferation and invasion in vitro (A) WB confirms the overexpression efficiency of TSPAN4 in U87, U251, and GBM1 cells. (B and C) CCK-8 and colony formation assays show the effect of TSPAN4 overexpression on cell proliferation in U87, U251, and GBM1 cells. (D) Transwell assay shows the effect of TSPAN4 overexpression on invasion in U87, U251, and GBM1 cells. Scale bar, 50 μm. (E and F) Representative images of EdU and Ki67 staining and the comparison of EdU-positive and Ki67-positive ratio upon TSPAN4 overexpression in U87 and U251 cells. Scale bar, 100 μm. Data in (B–F) are presented as the mean ± SD (n = 3). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. One-way ANOVA with Tukey’s test for B, Two-tailed Student’s t test for (C–F). TSPAN4 potentiates tumorigenicity of GBM in vivo To determine the tumorigenicity of TSPAN4 in vivo, we established an intracranial xenograft model in nude mice by transplanting U87-luciferase cells with stable infection of shCtrl, shTSPAN4-1#, and shTSPAN4-2# lentivirus. As expected, bioluminescence imaging (BLI) revealed a significant inhibition of intracranial tumor growth in vivo following TSPAN4 knockdown, compared to the control group ([91]Figures 4A and 4B). Additionally, tumor-bearing nude mice with TSPAN4 knockdown exhibited significantly greater body weight and overall survival ([92]Figures 4C and 4D). Hematoxylin-eosin (HE) staining of intracranial tumor tissues demonstrated tumor shrinkage upon TSPAN4 knockdown ([93]Figure 4E). Similarly, the IHC assay showed decreased Ki67 expression and increased active caspase-3 expression in the shTSPAN4 group compared to the control group, implying that TSPAN4 knockdown inhibited GBM cell proliferation and promoted apoptosis in vivo ([94]Figures 4F and 4G). All the previous findings demonstrate that TSPAN4 knockdown dramatically attenuates the tumorigenicity of GBM cells in vivo. Figure 4. [95]Figure 4 [96]Open in a new tab TSPAN4 knockdown attenuates tumorigenicity of GBM in vivo (A and B) Effect of TSPAN4 knockdown on brain tumor xenograft growth of U87-luciferase cells (A, representative images of bioluminescence imaging; B, quantification of bioluminescence imaging). (C and D) Effect of TSPAN4 knockdown on body weight and overall survival of tumor-bearing nude mice. (E) Representative images of HE of brain tissues of tumor-bearing nude mice. (F and G) Effect of TSPAN4 knockdown on the expression of TSPAN4, Ki67, and active caspase-3 in tumor-bearing nude mice (F, representative images of IHC staining; G, analysis of IHC scores of protein staining). Scale bar, 50 μm. Data in (B–D), and (G) are presented as the mean ± SD (n = 6). ^∗∗p < 0.01, ^∗∗∗p < 0.001. One-way ANOVA with Tukey’s test. Transcriptomic and proteomic changes upon TSPAN4 depletion in GBM cells To explore the mechanism underlying TSPAN4’s involvement in GBM tumorigenicity, we conducted transcriptomic and proteomic analyses following TSPAN4 knockdown in U87 cells ([97]Figure 5A). First, RNA sequencing (RNA-seq) and differentially expressed gene (DEG) analyses identified 919 upregulated and 971 downregulated TSPAN4-related DEGs, with a threshold of |log[2] FC|≥1 and adjusted p < 0.05 ([98]Figure 5B). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that TSPAN4-related upregulated DEGs were primarily connected to hypoxia and angiogenesis, while downregulated DEGs participated in cell cycle regulation ([99]Figure 5C). Furthermore, gene set enrichment analysis (GSEA) analysis revealed that TSPAN4 was associated with signaling pathways such as E2F targets, G2/M checkpoint, cell cycle, and DNA replication ([100]Figure 5D), implying a close association between TSPAN4 and cell cycle regulation. To validate the reliability of the bioinformatic analysis, qPCR was performed to assess the mRNA expression of CDK1, CDK2, and CCNB1, key regulators of the cell cycle. The findings revealed a significant decrease in the mRNA expression levels of CDK1, CDK2, and CCNB1 upon TSPAN4 knockdown in U87 and U251 cells ([101]Figure 5E). Figure 5. [102]Figure 5 [103]Open in a new tab TSPAN4 is involved in regulating the GBM cell cycle (A) Flowchart showing extraction of total RNA and proteins from shCtrl and shTSPAN4 groups in U87 and U251 cells for transcriptomics and proteomics analysis, respectively. (B) Volcano plot of TSPAN4-related differentially expressed genes (DEGs) in the shCtrl group versus the shTSPAN4 group in U87 cells. (C) GO and KEGG enrichment analyses of TSPAN4-related upregulated and downregulated DEGs. (D) GSEA pathway enrichment analysis of TSPAN4-related DEGs. (E) qPCR analysis of CDK1, CDK2, and CCNB1 mRNA level changes upon TSPAN4 depletion in U87 and U251 cells. (F) Volcano plot of TSPAN4-related differentially expressed proteins (DEPs) in the shCtrl group versus the shTSPAN4 group in U87 cells. (G) GO and KEGG enrichment analysis of TSPAN4-related up- and down-regulated DEPs. (H) WB confirms CDK1, CDK2, and CCNB1 protein level changes upon TSPAN4 depletion in U87 and U251 cells. Data in E are presented as the mean ± SD (n = 3). ^∗∗∗p < 0.001. One-way ANOVA with Tukey’s test. Subsequently, proteomic analysis revealed 536 upregulated and 814 downregulated differentially expressed proteins (DEPs) associated with TSPAN4 ([104]Figure 5F). Consistent with the previous findings, the downregulated DEPs related to TSPAN4 were primarily associated with the cell cycle, including processes such as DNA replication, cell division, DNA repair, and mismatch repair ([105]Figure 5G). Moreover, TSPAN4 knockdown notably reduced the protein levels of CDK1, CDK2, and CCNB1 in U87 and U251 cells ([106]Figure 5H). Collectively, these results indicate that TSPAN4 promotes GBM cell proliferation, in part, by regulating the expression of CDK1, CDK2, and CCNB1, thereby disrupted cell cycle homeostasis. TSPAN4 interacts with EGFR in GBM cells To further elucidate how TSPAN4 contributes to GBM progress, we conducted co-immunoprecipitation (coIP) to identify the interactome of TSPAN4. Initially, we generated U87 and U251 cells overexpressing TSPAN4-FLAG, followed by pull-down of TSPAN4-interacting proteins using anti-FLAG beads, and subsequently performed mass spectrometry (MS) analysis ([107]Figure 6A). Venn diagram revealed 128 overlapping TSPAN4-interacting proteins identified in both U87 and U251 cells ([108]Figure 6B). To characterize these TSPAN4-interacting proteins, GO analysis indicated that these proteins were primarily anchored to the membrane, governing protein secretion and transmembrane transporter activity ([109]Figure S3A). Specifically, we identified the top 6 potential TSPAN4-interacting proteins, namely GNAI2, RTN4, CANX, TMEM33, EGFR, and CD44 ([110]Figure 6C). Among these proteins, EGFR acts as a cell membrane receptor that receives signals from growth factors and transmits them into the cell, thereby regulating cell growth and apoptosis.[111]^17^,[112]^18^,[113]^19^,[114]^20 Additionally, aberrant expression of EGFR has been associated with the development and drug resistance of various tumors, including GBM.[115]^21^,[116]^22 Considering the crucial role of EGFR in GBM, we identified EGFR as a potential interacting protein for TSPAN4 for further investigation. Figure 6. [117]Figure 6 [118]Open in a new tab TSPAN4 interacts with EGFR in GBM cells (A) Schematic diagram showing the workflow to identify the interactome of TSPAN4 by coIP and mass spectrometry (MS). (B) Venn diagram showing overlapping proteins that potentially interacts with TSPAN4 in U87 and U251 cells. (C) The table shows the fold change of TSPAN4 with potentially interacting proteins according to PSM in the TSPAN4-FLAG group compared to the FLAG group. (D) Exogenous coIP of TSPAN4-FLAG and EGFR-HA from the transfected cell lysates in HEK293T cells. (E) Endogenous coIP of EGFR and TSPAN4 in U87 and U251 cells. (F) Im\munofluorescence shows colocalization between TSPAN4-GFP and EGFR-mCherry in U87 cells. Scale bar, 10 μm. The regions marked with white boxes are enlarged, and the fluorescence intensity along the dashed line is quantified. (G) Immunofluorescence shows colocalization between TSPAN4 and EGFR in U87, U251, and GBM1 cells. Scale bar, 30 μm. (H) TSPAN4-EGFR interaction domain prediction by HDOCK. The interaction between TSPAN4 (green) and EGFR (cyan) is depicted, with the protein structure displaying the interaction interface. (I) Schematic representation of the main domains of TSPAN4 and EGFR. (J) CoIP of different TSPAN4 truncations and EGFR-HA from the transfected cell lysates in U87 cells. (K) CoIP of TSPAN4-HA and different EGFR truncations from the transfected cell lysates in U87 cells. To verify the reliability of the previous MS results, we performed the coIP assay. Combined exogenous and endogenous coIP revealed that TSPAN4 indeed interacted with EGFR ([119]Figures 6D and 6E). Additionally, we overexpressed TSPAN4-GFP and EGFR-mCherry in U87 cells and conducted immunofluorescence staining to visualize their cellular localization. The results demonstrated clear expression of both proteins on the cell membrane, with significant co-localization observed in this compartment ([120]Figure 6F). Subsequently, we labeled endogenous TSPAN4 and EGFR in U87, U251, and GBM1 cells using immunofluorescence staining. Consistently, both proteins were primarily localized to the cell membrane, and co-localization was evident in this cellular compartment as well ([121]Figure 6G). The results aforementioned clearly show a significant association between TSPAN4 and EGFR. Based on the previous observations, we sought to identify which regions of TSPAN4 are critically required for its interaction with EGFR. Initially, we employed HDOCK analysis to predict that the interaction occurs within the extracellular region of TSPAN4 and EGFR ([122]Figures 6H and [123]S6C). HDOCK is a novel web server for protein-protein docking based on a hybrid strategy.[124]^23 To further validate their specific interaction domains, we conducted a series of truncation experiments of TSPAN4 and EGFR domains in U87 cells. The results demonstrated that the extracellular region spanning residues 105–202 of TSPAN4 and the extracellular region 480–620 of EGFR were essential for their interaction ([125]Figures 6I–6K). Collectively, our findings show that TSPAN4 indeed interacts with EGFR in GBM cells. TSPAN4 enhances EGFR protein stability in GBM cells To clarify the regulatory relationship between TSPAN4 and EGFR, we initially examined EGFR expression at both mRNA and protein levels following TSPAN4 knockdown. The results revealed that TSPAN4 knockdown in GBM cells diminished the protein level of EGFR at different time points, regardless of EGF treatment, while not affecting its mRNA expression ([126]Figures 7A, 7B, and [127]S4A). Moreover, analysis of the TCGA-GBM cohort revealed no significant difference in EGFR mRNA expression levels between the high and low TSPAN4 expression groups ([128]Figure S4B). Additionally, IHC staining of intracranial tumor tissues in nude mice revealed decreased EGFR protein levels following TSPAN4 knockdown ([129]Figure 7C). Furthermore, IHC staining of EGFR on GBM tissue microarrays demonstrated a significant increase in EGFR expression in GBM tissues compared to normal tissues ([130]Figures 7E and 7F). Importantly, we observed a positive correlation between EGFR expression and TSPAN4 expression levels ([131]Figure 7G). These findings imply that TSPAN4 can regulate EGFR at the post-transcriptional level, rather than at the transcriptional level. Figure 7. [132]Figure 7 [133]Open in a new tab TSPAN4 maintains the stability of EGFR protein (A and B) qPCR and WB analyses of changes in EGFR mRNA and protein levels after TSPAN4 knockdown in U87, U251, and GBM1 cells, respectively. (C and D) Effect of TSPAN4 knockdown on the expression of EGFR in tumor-bearing nude mice (C, representative images of IHC staining; D, analysis of IHC scores of EGFR staining). Scale bar, 50 μm. (E and F) IHC staining showing EGFR expression levels on a GBM tissue microarray. (E, representative images of IHC staining; F, analysis of IHC scores of EGFR staining in normal brain and GBM). Scale bar, 50 μm. (G) Scatterplot depicting the correlation between the H-score of TSPAN4 and EGFR in tissue microarray. (H) WB confirms alterations in EGFR expression after cycloheximide (CHX) treatment for 0, 4, 8, 12, and 24 h upon TSPAN4 depletion in U87 cells. (I) WB shows alterations in EGFR expression by MG132 or Baf treatment upon TSPAN4 knockdown in U87 and U251 cells. (J and K) WB shows changes in EGFR expression after transfection of TSPAN4-mut and TSPAN4- [MATH: Δ :MATH] 1–105 upon TSPAN4 depletion in U87 and U251 cells. (L and M) Effects of knockdown or overexpression of TSPAN4 in U87 and U251 cells on expression levels of EGFR and its downstream signaling pathways, including p-EGFR, p-MEK, p-ERK, p-STAT3, and p-AKT. Data in A, D, F, and H are presented as the mean ± SD, n = 3 for A and H, n = 6 for D. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ns., not significant. One-way ANOVA with Tukey’s test for A, D, and H. Two-tailed Student’s t test for F. To further elucidate the potential mechanism underlying TSPAN4-mediated EGFR stabilization, we assessed changes in EGFR protein expression following treatment with cycloheximide (CHX), MG132, and bafilomycin A1 (Baf). Initially, the CHX assay revealed that TSPAN4 knockdown in U87 and U251 cells significantly shortened the half-life of EGFR protein ([134]Figures 7H and [135]S4C). Subsequent treatment with the proteasome inhibitor MG132 restored EGFR expression, while treatment with Baf, a lysosome inhibitor, did not alter EGFR protein levels after TSPAN4 knockdown in U87 and U251 cells ([136]Figure 7I). These findings suggest that TSPAN4 knockdown destabilizes EGFR expression, primarily through the ubiquitination-proteasome pathway. To further confirm the crucial role of TSPAN4 in EGFR stability, we conducted TSPAN4 rescue experiments in U87 and U251 cells to assess the restoration of EGFR protein levels. The results showed that the reintroduction of TSPAN4 led to the recovery of EGFR levels in both cell lines ([137]Figure 7J). Additionally, we investigated the impact of the extracellular domain of TSPAN4 on EGFR expression. Our results demonstrated that under conditions of TSPAN4 knockdown, the reintroduction of the extracellular domain of TSPAN4 partially restored EGFR expression ([138]Figure 7K). Collectively, these findings indicate that TSPAN4 interacts with EGFR and stabilizes its expression. TSPAN4 exerts its oncogenic properties by activating the EGFR signaling pathway To investigate the effect of TSPAN4 on the downstream pathway of the EGFR, the WB assay confirmed that TSPAN4 knockdown reduced not only the protein level of EGFR but also the phosphorylation level of EGFR (p-EGFR). Sequentially, we observed that downstream levels of p-MEK, p-ERK, p-STAT3, and p-AKT were downregulated, while total level of MEK, ERK, STAT3, and AKT remained unchanged ([139]Figure 7L). In contrast, overexpression of TSPAN4 activated EGFR and its downstream signaling pathway ([140]Figure 7M). Previous studies have shown that the EGFR signaling pathway promotes cell proliferation by positively regulating key cell cycle-related genes, such as CDK1, CDK2, and CCNB1.[141]^24^,[142]^25^,[143]^26 Therefore, we hypothesized that TSPAN4 knockdown negatively regulated the expression of CDK1, CDK2, and CCNB1, leading to cycle G2/M phase arrest in GBM cell, possibly through inactivating EGFR signaling. To elucidate whether TSPAN4 promotes GBM cell proliferation and invasion through EGFR, we initially established TSPAN4 knockdown and EGFR overexpression in U87 and U251 cells ([144]Figure 8A). The results demonstrated that TSPAN4 knockdown remarkably attenuated the proliferation and invasion of GBM cells. Subsequent rescue experiments indicated that EGFR overexpression partially rescued the inhibitory effects of TSPAN4 knockdown on cell proliferation and invasion ([145]Figures 8B–8D). Moreover, co-treatment with TSPAN4 knockdown and EGFR inhibitors (Gefitinib) significantly suppressed the proliferation of U87 and U251 cells, suggesting a synergistic anticancer effect ([146]Figures 8E and 8F). These results suggest that TSPAN4 promotes GBM progression by modulating EGFR stability. Figure 8. [147]Figure 8 [148]Open in a new tab TSPAN4 modulates the tumorigenicity of GBM by influencing the EGFR signaling pathway (A) WB validates overexpression of EGFR efficiency upon TSPAN4 knockdown in U87 and U251 cells. (B–D) CCK-8, colony formation, and transwell assays show the effects of overexpression of EGFR on cell proliferation and invasion upon TSPAN4 knockdown in U87 and U251 cells, respectively. (E and F) CCK-8 assay shows the effect of TSPAN4 knockdown and EGFR inhibitors (Gefitinib) treatment on cell proliferation and invasion upon TSPAN4 knockdown in U87 and U251 cells, respectively. Data in B–F are presented as the mean ± SD (n = 3). ^∗∗∗p < 0.001. One-way ANOVA with Tukey’s test. Discussion Recent research has indicated that TSPAN4 is associated with tumor growth, metastasis, and drug resistance.[149]^10^,[150]^11^,[151]^12 However, the exact regulatory role of TSPAN4 in tumors remains largely unclear, particularly in GBM. Here, we demonstrate that TSPAN4 is highly expressed in gliomas and correlates with tumor progression and poor prognosis. TSPAN4 knockdown attenuates tumorigenicity of GBM both in vitro and in vivo. Furthermore, TSPAN4 directly interacts with EGFR and is required for EGFR stability. Through its regulation of the EGFR signaling pathway, TSPAN4 promotes GBM progression. Thus, our experimental evidence confirms that TSPAN4 functions as an oncogene in GBM, enhancing tumorigenicity by regulating EGFR stability. EGFR plays a pivotal role in the initiation and progression of GBM, with over 50% of GBM cases exhibiting EGFR amplification or mutation.[152]^27 These alterations in EGFR, including amplification and the emergence of EGFRvIII mutants, can dysregulate downstream signaling pathways, driving processes crucial for GBM pathogenesis.[153]^28 Consequently, targeting EGFR is considered the most promising therapeutic strategy for GBM. However, numerous clinical studies have indicated that GBM patients often display insensitivity to EGFR-targeted inhibitors, with limited clinical efficacy.[154]^29 One of the primary reasons for this challenge stems from the incomplete understanding of the mechanisms responsible for maintaining EGFR’s persistent membrane localization and the activation of its signaling pathway in GBM. With GBM’s notable intratumoral heterogeneity, therapies targeting a singular factor often provoke drug resistance. Therefore, acquiring a comprehensive understanding of the regulatory mechanisms governing EGFR in GBM holds promise for identifying novel drug targets and resolving the issue of EGFR drug resistance in GBM. Dysregulation of EGFR protein stability plays a critical role in abnormal EGFR signaling and cancer development.[155]^30^,[156]^31^,[157]^32 Therefore, understanding the molecular mechanisms controlling EGFR protein stability is essential. Our study identified TSPAN4 as an interactor with EGFR, enhancing EGFR stability and activating its downstream signaling pathway, thus promoting GBM progression. This contributes new insights into the complex regulation of EGFR, particularly post-translational modification. Nevertheless, future research needs to study in detail the mechanism of how TSPAN4 precisely regulates the stability of EGFR. We speculate that TSPAN4 may stabilize EGFR protein by recruiting EGFR deubiquitinase, and its interaction with EGFR may hinder E3 ligase binding, thereby promoting EGFR stability. Additionally, literature suggests that EGFR release from lipid rafts can activate EGFR. TSPAN4, as a membrane protein, may interact with lipid rafts to facilitate EGFR release and activation.[158]^33 In summary, our study unveiled that TSPAN4 promotes GBM progression by modulating EGFR stability and activating its downstream signaling pathways. TSPAN4 functions as an oncogene in GBM, and our findings shed light on the pivotal role of the TSPAN4-EGFR regulatory axis in GBM development. This discovery not only enhances our understanding of GBM pathogenesis but also identifies a novel therapeutic target to counteract EGFR resistance in GBM. Limitations of the study Our study suggests a direct interaction between TSPAN4 and EGFR on the plasma membrane. However, the specificity of this interaction was demonstrated without standard negative controls such as Na, K-ATPase, which raises concerns about our conclusions. Moreover, the truncation coIP experiments may not accurately represent the interaction dynamics of these proteins due to methodological limitations. Future studies should incorporate appropriate negative controls and consider a range of deletion constructs that more accurately reflect the structural and functional domains of these proteins. Furthermore, our study lacks a primary in vivo GBM model, which is essential for validating the therapeutic potential of targeting TSPAN4 in a clinically relevant setting. Our experiments primarily used cell line models, which, while valuable for dissecting molecular mechanisms, do not fully replicate the complexity and heterogeneity of human tumors. In vivo models derived from patient tumor samples would offer a more accurate reflection of the natural tumor environment, capturing interactions with the immune system, the influence of the microenvironment, and the architectural complexity of tumors. Consequently, further research is required to establish and utilize primary in vivo GBM models to enhance the translational value of our findings. STAR★Methods Key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies __________________________________________________________________ Rabbit polyclonal anti-TSPAN4 Novus Cat# NBP1-59438; RRID:[159]AB_11002719 Mouse monoclonal anti-FLAG Sigma Cat# F1804; RRID:[160]AB_262044 Mouse monoclonal anti-HA Sigma Cat# H9658; RRID:[161]AB_260092 Rabbit monoclonal anti-FLAG CST Cat# 14793; RRID:[162]AB_2572291 Rabbit monoclonal anti-HA CST Cat# 3724; RRID:[163]AB_1549585 Rabbit monoclonal anti-GAPDH CST Cat# 5174; RRID:[164]AB_10622025 Rabbit monoclonal anti-β-Tubulin CST Cat# 2128; RRID:[165]AB_823664 Rabbit monoclonal anti-CDK1 Abcam Cat# ab133327; RRID:[166]AB_11155333 Rabbit monoclonal anti-CDK2 Abcam Cat# ab32147; RRID:[167]AB_726775 Rabbit monoclonal anti-CCNB1 Abcam Cat# ab32053; RRID:[168]AB_731779 Rabbit monoclonal anti-cleaved Caspase-3 CST Cat# 9661; RRID:[169]AB_2341188 Rabbit polyclonal anti-Ki67 Abcam Cat# ab15580; RRID:[170]AB_443209 Rabbit monoclonal anti-EGFR Abcam Cat# ab52894; RRID:[171]AB_869579 Mouse monoclonal anti-EGFR Abcam Cat# ab30; RRID:[172]AB_303483 Mouse monoclonal anti-EGFR Proteintech Cat# 66455-1-Ig; RRID:[173]AB_2881824 Rabbit monoclonal anti-p-EGFR CST Cat# 3777; RRID:[174]AB_2096270 Rabbit monoclonal anti-MEK1/2 CST Cat# 9126; RRID:[175]AB_331778 Rabbit monoclonal anti-p-MEK1/2 CST Cat# 9154; RRID:[176]AB_2138017 Rabbit monoclonal anti-ERK1/2 CST Cat# 4695; RRID:[177]AB_390779 Rabbit monoclonal anti-p-ERK1/2 CST Cat# 4370; RRID:[178]AB_2315112 Rabbit monoclonal anti-AKT CST Cat# 4685; RRID:[179]AB_2225340 Rabbit monoclonal anti-p-AKT CST Cat# 4060; RRID:[180]AB_2315049 Rabbit monoclonal anti-STAT3 CST Cat# 12640; RRID:[181]AB_2629499 Rabbit monoclonal anti-p-STAT3 CST Cat# 9145; RRID:[182]AB_2491009 Goat polyclonal anti-mouse IgG (H + L) Secondary Antibody, HRP Thermo Fisher Scientific Cat#31430; RRID: [183]AB_228307 Goat polyclonal anti-rabbit IgG (H + L) Secondary Antibody, HRP Thermo Fisher Scientific Cat#31460; RRID: [184]AB_228341 Donkey anti-Rabbit Alexa Fluor 488 secondary antibody Thermo Fisher Scientific Cat# A-21206; RRID:[185]AB_2535792 Donkey anti-Rabbit Alexa Fluor 594 secondary antibody Thermo Fisher Scientific Cat# SA5-10040; RRID:[186]AB_2556620 __________________________________________________________________ Bacterial and virus strains __________________________________________________________________ TOP10 competent cell Tsingke TSC-C12 __________________________________________________________________ Biological samples __________________________________________________________________ GBM tissue microarrays Wuhan Servicebio IWLT-C-70GL61 __________________________________________________________________ Chemicals, peptides, and recombinant proteins __________________________________________________________________ Protease inhibitor cocktail Sigma Cat# P8340 Cycloheximide Sigma Cat# C4859 MG132 Sigma Cat# 474790 Bafilomycin A1 Sigma Cat# 196000 EGFR inhibitor (Gefitinib) Selleck Cat# S1025 Trizol reagent Invitrogen Cat# 15596018 Matrigel BD Cat# 356234 Lipofectamine 2000 Invitrogen Cat# 1668019 Recombinant Human EGF Protein R&D Cat# 236-EG Luciferin Promega Cat# P1041 Protein A/G beads Pierce Cat# 88803 Poly-L-lysine Sigma Cat# P6407 Prodidium Iodide (PI) Staining Solution BD Cat# 556463 Crystal Violet Staining Solution Beyotime Cat# C0121 Hoechst33342 staining solution NOVON MC160 __________________________________________________________________ Critical commercial assays __________________________________________________________________ EdU Cell Proliferation Kit Beyotime Cat# C0071S Cell Counting Kit-8 Beyotime Cat# C0040 Cell Cycle and Apoptosis Analysis Kit Beyotime Cat# C1052 SYBR Green Master Mix Yeasen Cat# 11203ES08 __________________________________________________________________ Deposited data __________________________________________________________________ RNA-seq and data This paper [187]https://dataview.ncbi.nlm.nih.gov/object/PRJNA1121048 GTEx database Open-source [188]https://www.gtexportal.org/ TCGA database (TCGA-LGG and TCGA-GBM) Open-source [189]https://portal.gdc.cancer.gov/ CGGA database Open-source [190]http://www.cgga.org.cn/ GEO database ([191]GSE16011 and [192]GSE107850) Open-source [193]http://www.ncbi.nlm.nih.gov/geo/ Original data of gels and blots This paper [194]https://data.mendeley.com/datasets/r7szjyfh9s/1 __________________________________________________________________ Experimental models: Cell lines __________________________________________________________________ HEK293T cell line ATCC RRID:CVCL_0063 U87 cell line ATCC RRID:CVCL_0022 U251 cell line ATCC RRID:CVCL_0021 Primary human GBM cells (GBM1) Procell Cat# CP-H148 __________________________________________________________________ Experimental models: Organisms/strains __________________________________________________________________ BALB/c nude mice Beijing Vital River Laboratory N/A __________________________________________________________________ Oligonucleotides __________________________________________________________________ TSPAN4 Forward: CTGCTTGGAGTTCAGTGAGAGC This paper N/A TSPAN4 Reverse: AGCAGGTTCTCCTGAAGCCACA This paper N/A CDK1 Forward: GGAAACCAGGAAGCCTAGCATC This paper N/A CDK1 Reverse: GGATGATTCAGTGCCATTTTGCC This paper N/A CDK2 Forward: ATGGATGCCTCTGCTCTCACTG This paper N/A CDK2 Reverse: CCCGATGAGAATGGCAGAAAGCC This paper N/A CCNB1 Forward: GACCTGTGTCAGGCTTTCTCTG This paper N/A CCNB1 Reverse: GGTATTTTGGTCTGACTGCTTGC This paper N/A EGFR Forward: AACACCCTGGTCTGGAAGTACG This paper N/A EGFR Reverse: TCGTTGGACAGCCTTCAAGACC This paper N/A GAPDH Forward: GTCTCCTCTGACTTCAACAGCG This paper N/A GAPDH Reverse: ACCACCCTGTTGCTGTAGCCAA This paper N/A shRNA targeting sequence: Ctrl: CAACAAGATGAAGAGCACCAA This paper N/A shRNA targeting sequence: TSPAN4-1#: CTCCAACTACACTGACTGGTT This paper N/A shRNA targeting sequence: TSPAN4-2#: GAGCATCATCCAGACCGACTT This paper N/A __________________________________________________________________ Recombinant DNA __________________________________________________________________ pLKO.1-shCtrl-hygro This paper N/A pLKO.1-shTSPAN4-hygro This paper N/A pcDNA3- HA-mCherry-tagged EGFR This paper N/A pcDNA3- FLAG-GFP-tagged TSPAN4 This paper N/A pcDNA3- FLAG-tagged TSPAN4 This paper N/A pcDNA3- FLAG-tagged EGFR This paper N/A __________________________________________________________________ Software and algorithms __________________________________________________________________ ImageJ NIH [195]https://imagej.nih.gov/ij/ R Studio The R foundation [196]https://www.r-project.org/ Adobe Illustrator Adobe [197]https://www.adobe.com/ GraphPad Prism 8 GraphPad Software [198]https://www.graphpad.com/ __________________________________________________________________ Other __________________________________________________________________ Laser scanning confocal focus microscope Olympus FV-1000 IVIS machine PerkinElmer IVIS® Lumina III [199]Open in a new tab Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the [200]lead contact, Yongshuo Liu (liuyongshuo@pku.edu.cn). Materials availability This study did not generate new unique reagents and all materials in this study are commercially available. Data and code availability * • The data reported in this paper are available in Deposited data in the [201]key resources table. * • This paper does not report original code. * • Any additional information required to reanalyze the data reported in this paper is available from the [202]lead contact upon request. Experimental model and study participant details Cell culture Cell lines U87, U251 and HEK293T were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin at 37°C in 5% CO2. The authenticated primary human GBM cells (GBM1), obtained from Wuhan Procell, was derived from GBM surgical specimens and maintained in primary serum-free cultures grown on laminin.[203]^34 Mice 6-week-old male BALB/c nude mice were purchased from Beijing Vital River Laboratory. Nude mice were maintained in the specific pathogen-free facility at Shandong Cancer Hospital and Institute. All animal experiments were approved by Ethics Committee of Shandong Cancer Hospital and Institute. Tissue microarray GBM tissue microarrays were purchased from Wuhan Servicebio, comprising 60 GBM and 8 normal brain tissue biopsies, accompanied by essential patient information ([204]Table S1). These arrays were utilized for immunohistochemical staining analysis of TSPAN4 and EGFR. All experimental procedures were approved by the Ethics Committee of Shandong Cancer Hospital and Institute. Method details Plasmid construction and lentiviral infection shCtrl and shTSPAN4 sequences were integrated into pLKO.1-hygro plasmid. FLAG-tagged TSPAN4, HA-tagged TSPAN4, FLAG-GFP-tagged TSPAN4, FLAG-tagged EGFR, HA-tagged EGFR, HA-mCherry-tagged EGFR, EGFR domain deletion mutants (EGFR-Δ1-273, Δ1-480, Δ1-620, Δ1-643, Δ1-954), TSPAN4 domain deletion mutants (TSPAN4-Δ1-105, Δ1-202) and TSPAN4 synonymous mutant (TSPAN4-mut) were integrated into pcDNA3.0 plasmid. For lentiviral infection, HEK293T cells were seeded into 10 cm dishes. The cells were transfected with pMD2.G, psPAX2, and either pLKO.1 or pLenti-CMV plasmids using Lipofectamine 2000. Lentivirus was collected at 48 h. Cells were infected with the lentivirus for 48 h and then selected with antibiotics for 3 days. RNA isolation and qPCR Total RNA was extracted using Trizol reagent and reverse transcribed using M-MLV Reverse Transcriptase. Quantitative real-time PCR was performed in triplicate using qPCR SYBR Green Master Mix. Western blot (WB) and Co-immunoprecipitation (Co-IP) Proteins were extracted using 1× SDS sample buffer (25 mM Tris-HCl, pH 6.8, 1% SDS, 5% Glycerol) supplemented with protease inhibitor cocktail. The protein samples were quantified using Nanodrop and standardized to equal concentrations. Subsequently, the protein samples were separated by 12% SDS-PAGE and transferred onto PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk and then incubated with the designated antibodies at 4°C overnight. The membranes were washed three times with TBST buffer and incubated with secondary antibodies for 1 h at room temperature. Afterward, the membranes were visualized using enhanced chemiluminescence reagents, and the signals were captured by film exposure.[205]^35 For exogenous Co-IP, cells were transfected with indicated plasmids. After 48 h of transfection, the cells were lysed using RIPA lysis buffer containing 1 × protease inhibitor cocktail and 1 × phosphatase inhibitor cocktail. For endogenous Co-IP, U87 and U251 cells in 10 cm dishes were directly lysed using lysis buffer. The cell lysates were subjected to immunoprecipitation using primary antibodies at 4°C overnight. Protein A/G beads were added to the lysates and rotated for 4 h at 4°C. Afterward, the beads were washed three times with lysis buffer and the immunoprecipitated proteins were eluted using 1 × LDS sample buffer and collected for immunoblotting. Cell proliferation, invasion, and cell cycle assays Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8) according to the manufacturer’s protocol. The absorbance at 450 nm was measured at indicated time points. For the invasion assay, 5 × 10^4 cells were placed into Transwell inserts with 8 μm pore size with precoated matrigel. After 24 h incubation, the invasive cells were stained with 0.1% crystal violet, observed under a microscope, and counted using Image J software. For cell cycle assay, 1×10^5 cells were stained with propidium iodide (PI) staining solution and subsequently analyzed using flow cytometry. Colony formation assay 1×10^3 cells were plated into 6-well plates and incubated for 14 days. Following incubation, the cells were fixed using 4% paraformaldehyde solution and then stained with 0.1% crystal violet. EdU and Ki67 staining For the EdU staining assay, 5×10^4 cells were seeded into coverslips pre-coated with Poly-L-lysine in 12-well plates. Subsequently, they were labeled with 10 μM EdU, fixed with 4% paraformaldehyde, permeabilized in 0.3% Triton X-100, and incubated in click-reaction reagent. The nucleus was stained with Hoechst 33342 staining solution. For the Ki67 staining assay, 5×10^4 cells were seeded into coverslips, followed by 4% PFA immobilization, 0.3% Triton X-100 permeabilization, and 5% BSA blocking. Cells were subsequently incubated with Ki67 primary antibody at 4°C overnight followed by Donkey anti-Rabbit Alexa Fluor 488 secondary antibody incubation for 1 h. The nucleus was stained with Hoechst 33342 for 10 min. The slides were mounted and photographed by a laser scanning confocal focus microscope. Immunohistochemistry staining The mouse brain tumor slides were incubated with TSPAN4, Ki67, active caspase-3, and EGFR antibody. Visualization was achieved through diaminobenzidine treatment followed by hematoxylin counterstain. The IHC score (H-score) is assessed by multiplying the intensity of positive staining (0-negative, 1-weak, 2-moderate, and 3-strong) with the percentage of stained area and the H-score values range from 0 to 300. RNA sequencing analysis Total RNA was isolated using Trizol reagent. Following digestion, purification, and reverse transcription, the resulting fragments were ligated with Illumina sequencing adapters. For mRNA expression analysis, the data were aligned to the reference genome using TopHat2 (v.2.1.1), and differentially expressed genes were identified using the DESeq2 software (v.1.24.0). The Gene Set Enrichment Analysis (GSEA) of differentially expressed genes was conducted using GSEA software. Liquid chromatography–MS/MS analysis Total protein was extracted using lysis buffer and separated on a 12% SDS-PAGE gel. The band was excised and subjected to enzymatic digestion. The resulting peptides were then analyzed using a QExactive mass spectrometer coupled to a nano-LC system (AdvanceLC). The acquired spectra were processed and interpreted using the SEQUEST HT algorithm. Xenograft assay 6-week-old male Balb/c nude mice were purchased from Beijing Vital River Laboratory. 1 × 10^6 U87-luciferase cells were slowly intracranially injected into the brains of nude mice. The approval of animal experimental procedures was obtained from the Shandong Cancer Hospital and Institute. Tumor growth was tracked using bioluminescence imaging (BLI). The mice were kept until the onset of neurological symptoms. BLI images were acquired and processed using living image software. TSPAN4 expression and prognostic value in glioma The public data used in this study are detailed in Deposited data in the [206]key resources table. We first compared the mRNA expression differences of TSPAN4 in normal, LGG, and GBM tissues using the TCGA, CGGA, and [207]GSE16011 databases, respectively. The relationship between TSPAN4 and clinicopathological characteristics of glioma patients was investigated using the TCGA database. The prognostic value of TSPAN4 was analyzed using data from the TCGA, CGGA, and [208]GSE107850 databases, respectively. In detail, Kaplan-Meier survival curves were conducted by the R package “survival” and visualized by the R package “ggplot2” and the R package “survminer”. Time-dependent ROC curves were analyzed by the R package “timeROC” and visualized by the R package “ggplot2”. Univariate Cox regression analysis was performed with the R package “survival” and forest plot was visualized with the R package “ggplot2”.[209]^36^,[210]^37 Quantification and statistical analysis All statistical analyses were processed on R Studio or GraphPad Prism 8 software, and p value <0.05 was considered statistically significant. The quantitative results are presented as the mean ± standard deviation (SD). Univariate Cox regression analysis and log rank test were performed to evaluate the prognostic value of TSPAN4 in glioma. Acknowledgments