| Despite of great progresses made in treatment of varieties of cancers in recent years,glioma remains still as one of the most malignant tumor. The benefit and improvement by current standard-of-cares including surgery, chemotherapy, and radiation on glioma is limiting.In 2016, cancer statistics from CA Cancer J Clin, reported that malignant brain tumors become leading cause for cancer death of children and adolescents. Therefore, the need for new drug or methods on glioma is urgent.Synthesis of novel NDGA-derived NDGA-P21 and identification of its anti-glioma effectNDGA, a natural compound extracted from creosote bush Larrea tridentate, is originally used as anti-oxidant in commercial foods in 1970s. Until late 1980s, NDGA is found capable of suppressing tumor growth and angiogenesis, in addition to its functional oxidation-resistance against UV damage and virus infection. In this current project, we synthetized a new derivative-NDGAP21 based on NDGA structure. Regardless of the structural similarity, NDGA-P21 exhibited stronger capability in suppression of glioblastoma(GBM) cell growth as compared to Nordy. Mechanically, NDGA-P21 is able to arrest cell cycle of GBM cells in G0/G1 phase, and to block cell proliferation sequentially. It is important to note that NDGA-P21 is able to impair the stemness of glioma stem-like cells(GSLCs) via measurement of colony formation and sphere formation. Taken together, the novel NDGA-based compound NDGA-P21 exhibits potential therapeutic implications through inhibiting proliferation of glioma cells and self-renewal capability of GSLCs.Identification of CXCR5+ PD-1+ CD4~+ T (Tfh-like) cells in human gliomaCarcinogenesis and tumor progression is closely related to tumor microenvironment(TM).TM is constituted with large numbers of immune-suppression components, which thus facilitate tumor cell immune evasion. Akin to other solid tumors, glioma TM is complexed,and comprising of varieties of cell types including tumor cells, stroma cells, cancer-associated fibroblast, tumor associated macrophage, dendritic cell, and lymphocyte, etc. Here, by focusing on analysis of tumor-infiltrating T cells, we identified a subset of CXCR5+ PD-1 ＋CD4~+ T (Tfh-like) cell in human glioma biopsy. Unlike breast-cancer- or colon-cancer-infiltrating Tfh-like cells, these glioma-infilatrating Tfh-like cells are negatively correlated with patients’ survival. Gene profiling demonstrates that glioma infiltrating Tfh-like cells are able to generate IL-8 in a great amount. Hence, our primary observation implicates a potential target for immune therapy of glioma.The main results and conclusions of the paper are as follows:1. Inhibitory effect of small molecule drug NDGA-P21 on glioma(1) After administration of different concentration of NDGA-P21 and Nordy on glioma cells in vitro for 72 hours, we counted the cell number, calculated the IC50 value of two drugs,on U87-MG cells: NDGA-P21 - 7.43 mol/L to, Nordy - 34.52 mol/L; while on primary glioma GBM1 cells: NDGA-P21 - 55.54 mol/L Nordy - 15.38 mol/L. Then U87-MG and GBM1 were observed for cell proliferation with treatment of DMSO, NDGA-P21 and Nordy.Then we ploted growth curve of glioma cells after cell counting, and found that compared to DMSO control group, NDGA-P21 and Nordy can significantly inhibit the proliferation of glioma cells (P<0.05).(2) Analysis of cell cycle and apoptosis by flow cytometry showed that NDGA-P21 could block glioma cells in G0/G1 phase, but had no significant effect on apoptosis.NDGA-P21 could significantly inhibit the expression of CCNE1, CDK2 and CDK6 in the G1-S phase transition, and up-regulate the expression of the inhibitory factor GADD45a in the G2-M phase at mRNA level.(3) NDGA-P21 could significantly inhibit the sphere formation and soft agar clone formation of glioma-stem-like cells(P<0.05), and inhibit the stem cell transcription factor Nanog mRNA expression,indicating that NDGA-P21 has the ability to inhibit self-renewal of glioma-stem -like cells.(4) Immunofluorescence staining showed that NDGA-P21 could induce the differentiation of glioma-stem-like cells to express GFAP, which indicate that NDGA-P21 could induce the differentiation of glioma-stem-like cells.2. Follicular helper T cell (Tfh) - like T cell infiltration in glioma microenvironment(1) With immunofluorescence analysis and flow cytometry analysis of lymphocytes infiltrated into tumor tissue in glioma patients,we found a group of local glioma tissue specific T cells with high expression of CXCR5 and PD-1, which are regarded as the molecular marker of Tfh cells in secondary lymphoid structure.(2) Through the comparison of infiltrating T cells in glioma tissue and T cells in peripheral blood, we found the high expression of CXCR5 and PD-1 in T cells is tissue-specific, T cells in peripheral blood only high express CXCR5, but not PD-1.(3) Glioma tissue and peripheral blood samples from same patient were collected for the analysis of transcriptome sequencing. Tumor tissue specific Tfh-like T cells had a specific gene transcription pattern. Through analyzing information of glioma patients in TCGA database with high accumulation of tumor specific Tfh-like T cells (characterized as expression of CD3D,CD4, CXCR5, PD-1,IL1B,C1QB,FCER1Q IL1RN,FOSB,IL8,TREM2 CSF1, CCL3), a negative correlation is found between the prognosis of patients with glioma and Tfh like T cells.(4) We constructed an orthotopic glioma xenograft model to test T lymphocytes infiltration,and found that similar to human samples,there are also Tfh-like T cells in glioma tissue.(5) In Bcl6 condition knocknout mice, the growth of glioma was inhibited, and the expression of immune suppressing marker Tim-3 in T cells was decreased.The main conclusions of this study are: NDGA derivative NDGA-P21 has a direct inhibitory effect on proliferation of glioma cells and inhibition of the self-renewal,that makes NDGA-P21 to showe good prospects for treatment of glioma. There are specific Tfh like CD4~+T cells in the microenvironment of glioma, and the degree of tumor specific Tfh like T cell enrichment is negatively correlated with the prognosis of glioma patients. |
PDF Full Text Request