Tumor recurrence, the chief reason for poor prognosis of glioma, is largely attributed to glioma stem cells (GSCs) and epithelial-mesenchymal transition (EMT). However, the mechanisms among them remain unknown. Here, we determined whether leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), known as a stem cell marker for colon cancer and gastric cancer, can serve as a novel GSC marker involved in EMT and a therapeutic target in glioma. Stemness properties were examined in FACS-isolated LGR5⁺/LGR5⁻ cells. Reported stem cell markers, EMT and the Wnt/β-catenin pathway were examined in stable LGR5 knockdown or overexpressed GSCs by Western Blot. The treatment experiment was performed in an intracranial orthotopic xenograft model by knockdown of LGR5 or by using the Wnt/β-catenin pathway inhibitor Wnt-C59. LGR5 expression was determined in 268 glioma specimens by immunohistochemistry. LGR5⁺ cells possessed stronger stemness properties compared to LGR5⁻ cells. The expression of SOX2, Nanog, CD133, CD44, CD24 and EpCAM was modulated by LGR5. Both LGR5 knockdown and Wnt-C59 reduced tumor invasion and migration and blocked EMT by inhibiting the Wnt/β-catenin pathway in vitro and suppressed the intracranial orthotopic xenograft growth and prolonged the survival of xenograft mice in vivo. Moreover, LGR5 was positively correlated with Ki67, N-cadherin and WHO grade and negatively correlated with IDH1. Glioma patients with high expression of LGR5 showed significantly poorer prognosis. LGR5 is a new functional GSC marker and prognostic indicator that can promote EMT by activating the Wnt/β-catenin pathway and would thus be a novel therapeutic target for glioma.

Background: Although the availability of therapeutic options including temozolomide, radiotherapy and some target agents following neurosurgery, the prognosis of glioma patients remains poor. Thus, there is an urgent need to explore possible targets for clinical treatment of this disease. Methods: Tissue microarrays and immunohistochemistry were used to detect FKBP10, Hsp47, p-AKT (Ser473), p-CREB (Ser133) and PCNA expression in glioma tissues and xenografts. CCK-8 tests, colony formation assays and xenograft model were performed to test proliferation ability of FKBP10 in glioma cells in vitro and in vivo. Quantitative reverse transcriptase-PCR, western-blotting, GST-pull down, co-immunoprecipitation and confocal-immunofluorescence staining assay were used to explore the molecular mechanism underlying the functions of overexpressed FKBP10 in glioma cells. Results: FKBP10 was highly expressed in glioma tissues and its expression was positively correlates with grade, poor prognosis. FKBP10-knockdown suppressed glioma cell proliferation in vitro and subcutaneous/orthotopic xenograft tumor growth in vivo. Silencing of FKBP10 reduced p-AKT (Ser473), p-CREB (Ser133), PCNA mRNA and PCNA protein expression in glioma cells. FKBP10 interacting with Hsp47 enhanced the proliferation ability of glioma cells via AKT-CREB-PCNA cascade. In addition, correlation between these molecules were also found in xenograft tumor and glioma tissues. Conclusions: We showed for the first time that FKBP10 is overexpressed in glioma and involved in proliferation of glioma cells by interacting with Hsp47 and activating AKT-CREB-PCNA signaling pathways. Our findings suggest that inhibition of FKBP10 related signaling might offer a potential therapeutic option for glioma patients.

Introduction: The prognosis of patients with glioma is dismal. It has been reported that Serpin peptidase inhibitor clade A member 3 (SERPINA3) is associated with the mobility and invasion of tumor cells. Our study was designed to explore the value of SERPINA3 messenger RNA (mRNA) expression in the biological process, prognosis, and immune significance in glioma.Methods: We analyzed the biological functions of SERPINA3 through data from the Chinese Glioma Genome Atlas databases. Differentially expressed genes and enrichment analysis were performed and correlations between SERPINA3 expression and immune cell infiltration were analyzed. Further, we validated the expression and the survival prediction role of SERPINA3 by using tissue microarrays and RNAscope in situ hybridization in 321 gliomas. The correlations between the expression and clinical‐pathological parameters as well as other biomarkers were examined.Results: Univariate and multivariate regression both indicated that the level of SERPINA3 transcript represented an independent prognostic factor. High levels of SERPINA3 correlated with poor survival in patients with glioma. Expression of SERPINA3 mRNA was observed positively correlated with MCM6, IGFBP2, and FKBP10. Enrichment analysis showed SERPINA3 mainly enriched in immune‐related terms and signaling pathways including MAPK, TNF, P53, PI3K‐Akt, nuclear factor‐κB. Immune infiltration analysis further declare the SERPINA3 expression negatively correlated with levels of Macrophages M1, native CD4⁺ T cell, monocytes, and Mast cell activated. And overexpression of SERPINA3 correlated with low CD4⁺ T cell infiltration in glioma tissues.Conclusions: SERPINA3 may play a key role in the biological process of glioma cells especially in immune suppression activities. SERPINA3 may serve as an independent survival prediction factor in glioma patients.

Hypothetically, intratumoral genomic heterogeneity has the potential to foster tumor-infiltrating lymphocyte (TIL) diversity; however, no study has directly tested this hypothesis by simultaneously investigating somatic mutations, TIL diversity, and immune response activity. Thus, we performed whole-exome sequencing, immune repertoire sequencing and gene expression on ten spatially separated tumor samples obtained from two tumor masses excised from a glioblastoma multiforme (GBM) patient, and we included peripheral blood as control. We found that although the multi-region samples from one tumor shared more common mutations than those from different tumors, the TIL populations did not. TIL repertoire diversity did not significantly correlate with the number of non-synonymous mutations; however, TIL diversity was highly correlated with local immune activity, as the pathways were all immune-related pathways that highly positive correlated with local TIL diversity. Twenty-three genes with expression largely unaffected by the intratumor heterogeneity were extracted from these pathways. Fifty GBM patients were stratified into two clusters by the expression of these genes with significant difference in prognosis. This finding was validated by The Cancer Genome Atlas (TCGA) GBM dataset, which indicated that despite the heterogeneity of intra-tumor immune status, the overall level of the immune response in GBM could be connected with prognosis.

The prognosis of patients with glioblastoma (GBM) is dismal. It has been reported that Insulin-like growth factor (IGF) binding protein 2 (IGFBP2) is associated with the mobility and invasion of tumor cells. We investigated the expression of IGFBP2 mRNA in GBMs and its clinical relevance, using tissue microarrays and RNAscope in situ hybridization in 180 GBMs and 13 normal or edematous tissues. The correlations between the expression and clinical pathological parameters as well as some other biomarkers were analyzed. Overexpression of IGFBP2 mRNA was observed in 23.9% of tumors tested. No expression of IGFBP2 mRNA was detected in normal or edematous tissues. Kaplan–Meier survival analysis showed that the survival time of all the patients with high IGFBP2 tumors had shorter survival than those with low IGFBP2 (P<0.01). Univariate regression and multivariate regression both indicated that the expression of IGFBP2 transcript level was an independent prognostic factor (P=0.008 and 0.007, respectively). Furthermore, expression of IGFBP2 mRNA was related to the occurrence of isocitrate dehydrogenase 1 (IDH1) mutation, high heat shock protein 27 (Hsp27) expression and telomerase reverse transcriptase (TERT) promoter mutation (TERTp⁺) (P=0.013, 0.015 and 0.016, respectively), and patients with TERTp⁺/IGFBP2^high showed the shortest survival. In conclusion, IGFBP2 mRNA expression status is an independent prognostic biomarker in GBMs, and the combination of IGFBP2 mRNA and TERTp status might serve as a prognostic indicator in patients with GBM.