MAGED4B Promotes Glioma Progression via Inactivation of the TNF-α-induced Apoptotic Pathway by Down-regulating TRIM27 Expression / 神经科学通报·英文版

MAGED4B belongs to the melanoma-associated antigen family; originally found in melanoma, it is expressed in various types of cancer, and is especially enriched in glioblastoma. However, the functional role and molecular mechanisms of MAGED4B in glioma are still unclear. In this study, we found that the MAGED4B level was higher in glioma tissue than that in non-cancer tissue, and the level was positively correlated with glioma grade, tumor diameter, Ki-67 level, and patient age. The patients with higher levels had a worse prognosis than those with lower MAGED4B levels. In glioma cells, MAGED4B overexpression promoted proliferation, invasion, and migration, as well as decreasing apoptosis and the chemosensitivity to cisplatin and temozolomide. On the contrary, MAGED4B knockdown in glioma cells inhibited proliferation, invasion, and migration, as well as increasing apoptosis and the chemosensitivity to cisplatin and temozolomide. MAGED4B knockdown also inhibited the growth of gliomas implanted into the rat brain. The interaction between MAGED4B and tripartite motif-containing 27 (TRIM27) in glioma cells was detected by co-immunoprecipitation assay, which showed that MAGED4B was co-localized with TRIM27. In addition, MAGED4B overexpression down-regulated the TRIM27 protein level, and this was blocked by carbobenzoxyl-L-leucyl-L-leucyl-L-leucine (MG132), an inhibitor of the proteasome. On the contrary, MAGED4B knockdown up-regulated the TRIM27 level. Furthermore, MAGED4B overexpression increased TRIM27 ubiquitination in the presence of MG132. Accordingly, MAGED4B down-regulated the protein levels of genes downstream of ubiquitin-specific protease 7 (USP7) involved in the tumor necrosis factor-alpha (TNF-α)-induced apoptotic pathway. These findings indicate that MAGED4B promotes glioma growth via a TRIM27/USP7/receptor-interacting serine/threonine-protein kinase 1 (RIP1)-dependent TNF-α-induced apoptotic pathway, which suggests that MAGED4B is a potential target for glioma diagnosis and treatment.

Glioma stem cell clones and molecular genetics characteristics of primary and recurrent gliomas / 中华神经医学杂志

Objective To explore the molecular genetic characteristics of primary and recurrent glioblastomas (GBMs) from the same patient in vivo, primary glioma stem cells cultured in vitro, and patient-derived xenograft (PDX). Methods (1) The primary and recurrent GBM specimens from one patient during surgical resection were collected; and the expressions of glial fibrillary acidic protein (GFAP), nestin and Ki-67 were detected by immunohistochemical staining; the methylation of O6-methylguanine DNA methyltransferase (MGMT) gene, mutation of isocitrate dehydrogenase (IDH) gene and amplification of epidermal growth factor receptor (EGFR) gene were analyzed. (2) The primary and recurrent GBM stem cells were cultured in vitro and named as SU5-1 and SU5-2 cells, respectively; the expressions of nestin and CD133 were detected by immunohistochemical staining; GFAP expression was detected by immunohistochemical staining after induced differentiation, and the growth curve was detected by CCK-8 assay; Transwell invasion assay was used to detect the invasion ability; cell resistance to temozolomide (TMZ), carboplatin (CBP), cisplatin (DDP) and adriamycin (ADM) was detected by CCK-8 assay; the protein expression of programmed death receptor-ligand 1 (PD-L1) was detected by Western blotting. The rate of PD-L1 positive cells was detected by flow cytometry; genetic testing analysis was as above. (3) The primary and recurrent in situ PDX models in nude mice were established, and the expressions of nestin, GFAP and Ki-67 were detected by immunohistochemical staining. Results (1) As compared with the primary GBM, the recurrent GBM had significantly higher percentages of Ki-67 and nestin positive cells, while statistically lower percentage of GFAP positive cells (P<0.05); genetic analysis showed that there was no mutation in IDH gene in the primary GBM tissues and recurrent GBM tissues; the MGMT gene in the primary GBM tissues was methylated and EGFR gene was not amplified, while the MGMT gene in recurrent GBM tissues was demethylated and EGFR gene amplification was positive. (2) Both SU5-1 and SU5-2 cells expressed nestin and CD133, and GFAP was expressed after induced differentiation; the growth curve showed that the proliferation of SU5-2 cells started earlier than that of SU5-1 cells, the two were equal on the 3rd, 4th, and 5th d, and the proliferation of SU5-1 cells was faster than that of SU5-2 cells from the 6th d; the invasion ability of SU5-2 cells was statistically stronger than that of SU5-1 cells (P<0.05); the inhibition rates of SU5-2 cells treated with 5, 10, and 15 mmol/L CBP, 0.3125, 1.25, and 5 mmol/L DDP, 0.5 and 2 mmol/L ADM, and 125 and 500 mmol/L TMZ were significantly lower than those of SU5-1 cells treated with the same concentrations and same drugs (P<0.05); the protein expression of PD-L1 in SU5-2 cells was higher than that in SU5-1 cells; the positive rate of PD-L1 in SU5-2 cells was statistically higher than that in SU5-1 cells (P<0.05); the results of genetic analysis were consistent with those of the primary and recurrent GBM samples. (3) As compared with those in the primary PDX model, the nestin and Ki-67 expressions were significantly higher and GFAP expression was significantly lower in the recurrent PDX model (P<0.05); the results of genetic analysis were consistent with those of the primary and recurrent GBM samples. Conclusions Genetic differences are detected between primary and recurrent GBMs; recurrent GBM has stronger invasive capacity and multi-drug resistance. The primary stem cells derived from surgical specimens and corresponding PDX models could replicate the molecular genetic characteristics of original tumors, which provide a reliable experimental platform for both tumor translation researches and screening of molecular therapeutic targets.

Establishment of a new congenic inbred mouse strain named Foxn1nu.B6-CAG-EGFP/SU / 中国比较医学杂志

Objective To establish a new congenic inbred mouse strain carrying and expressing EGFP and Foxn1nugene for cancer research including human glioma as well .Methods According to criterion of GB14923-2010, the male Foxn1nu nude mice backcross the female C57BL/6-Tg (CAG-EGFP) transgenic mice for 10 times, Then identify the phenotype using the methods and equipment as below: fluorescent flashlight and matching glasses; multifunction vivo imager; fluorescence microscopy.Results The congenic inbred mouse strain named Foxn1nu.B6-CAG-EGFP/SU ( Soochow University ) .All the 14 biochemical loci are homozygous and same with Balb/c mouse in addition to the Pep3 loci (“b” type instead of “a” type).Peripheral blood lymphocyte count shows the lymphocytes occupy 15%of nucleated cells;T lymphocytes occupy 0.3%, meet the requirement of inbred strain of EGFP nude mice .Conclusions Established a new congenic inbred strain -Foxn1nu.B6-CAG-EGFP/SU which both express EGFP stably, and own immunodeficiency with lack of T lymphocytes .The phenotype “b” of biochemical loci “Pep3” is the unique characteristic that distinguish SU to Foxn1nu.

Tumor angiogenesis promoted by fusion of glioma stem/progenitor cells with bone marrow mesenchymal stem cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>The aim of this study was to clarify whether the fusion of bone marrow mesenchymal stem cells (MSCs) with tumor cells can promote tumor angiogensis.</p><p><b>METHODS</b>Human glioma stem/progenitor cells (GSPCs) (SU3 cells) were transfected with red fluorescent protein (RFP) gene. Bone marrow mesenchymal stem cells (MSCs) were harvested from nude mice with whole-body green fluorescent protein (GFP) gene expression. Then the two kinds of cells were co-cultured in vitro. At the same time SU3-RFP was transplanted into the brain of GFP-expressing nude mice to establish xenograft tumors. The co-cultured cells, GFP/RFP double positive (yellow) cells and blood vessels obtained from the xenograft tumors were observed under fluorescent microscope and laser scanning confocal microscope.</p><p><b>RESULTS</b>After five passages in vitro, MSCs maintained the proliferative activity and highly expressed CD105. CD105 was also expressed in the femurs of GFP-expressing nude mice, tumor cells, blood vessels of SU3 xenograft tumors, and clinical malignant gliomas. When MSCs were co-cultured with SU3-RFP, the ratio of yellow cells co-expressing RFP and GFP was significantly increased after extended time and continuous passages. According to the flow cytometry, yellow cells co-expressing RFP and GFP were 83.7% of the cultured cells. In tissue slices of the xenograft tumors, bundles of yellow vessel-like structure and cross-sectioned yellow vascular wall structures including vascular wall stroma cells were observed with RFP and GFP expression, and were identified as de novo formed vessels derived from fusion of MSCs with SU3-RFP cells.</p><p><b>CONCLUSION</b>Cell fusion occurs between tumor cells and host MSCs and it promotes tumor angiogenesis.</p>

Malignant transformation of fusion cells derived from co-culturing human glioma cell line SU3 with murine macrophages / 中华微生物学和免疫学杂志

Objective To investigate whether the malignant transformation of macrophages ( Mφ) in glioma mesenchyme was induced by the fusion of glioma cells ( SU3 ) and Mφ.Methods SU3 cells transfected with red fluorescent protein genes were co-cultured in vitro with Mφexpressing enhanced green fluorescent protein.The cell lineages with RFP+/GFP+dual-color fluorescence were established by using monoclonal selection method.A series of tests for analyzing cancer-related phenotypes, tumorigenicity and specific markers for murine macrophage were performed.Results (1) A few of dual-color fluorescent cells were observed in the co-culture.Three monoclonal cell lineages (C3, C4 and C12) were obtained success-fully.(2) Three types of cells including RFP+, EGFP+and RFP+/EGFP+cells were formed during the cul-ture of monoclonal C12 cell lineage.The percentage of EGFP+cells was increased along the extended culture time and increased passages.Then, EGFP+cells gradually became the predominant cell population.Nota-bly, the percentage of RFP+/EGFP+cells were decreased and maintained at a low level, but the RFP+cells almost disappeared.(3) EGFP+cells from monoclonal C12 cell lineage showed the malignant characteristics such as loss of contact inhibition, rapid proliferation andchromosome aneuploidy, as well as high tumorigenic rate in nude mice (5/5).They also expressed macrophage specific marker CD68 and showed a large number of telocentric chromosomes.Conclusion The results of this study suggested that the malignant transforma-tion of host macrophages as previously observed in solid tumor might be induced by cell fusion occurred be-tween human glioma cells and macrophages.Along with the previous evidences showing the isolation of the malignantly transformed macrophages ( ihCTC) from solid tumor tissue of tumor-bearing mice, the results confirmed an objective existence of malignant transformation of host macrophages in tumor microenvironment. The malignant transformation of host cells induced by fusion with tumor cells revealed not only a new under-standing for the progression of tumor and cancer heterogeneity, but also new targets for cancer therapy.

Establishment and characterization of dual-color fluorescence nude mouse models of glioma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To establish red-green dual-color fluorescence glioma model in nude mice and to explore its practical values.</p><p><b>METHODS</b>CM-DiI-stained rat glioma C6 cells (C6-CM- DiI cells) expressing red fluorescence were inoculated into the brain of athymic nude mice expressing green fluorescence protein (NC-C57BL/6J-EGFP). Then the whole-body dual-color fluorescence imaging was detected dynamically. Finally whole brains of the tumor-bearing mice were removed and 5 µm thick serial frozen slices were made. Light microscopy, fluorescence microscopy and confocal laser scanning microscopy were performed to observe the transplanted tumor tissue structure and fluorescent cells.</p><p><b>RESULTS</b>Tumor mass with red fluorescence increased gradually under continuous in-vivo fluorescence imaging monitoring. Under the fluorescence microscope, cells with red, green and yellow fluorescence were observed in the frozen sections of transplanted tumor tissue and the mutual structural relationship among them could be defined. The tumor cells migration, implantation and cell fusion between transplanted tumor cells and host cells could be observed. It could be distinguished according to the fluorescence, that blood vessels of tumor-origin displayed red fluorescence, blood vessels of host-origin displayed green fluorescence and mosaic blood vessels appeared yellow fluorescence. It was depicted that host innate astrocytes and oligodendrocytes in the microenvironment at the tumor periphery could be activated and dedifferentiated into nestin-positive cells.</p><p><b>CONCLUSIONS</b>In contrast to traditional animal model, the dual-color fluorescence imaging of nude mouse models of glioma possesses enormous advantages in investigating tumor mass in-vivo fluorescence imaging, tumor cells migration and metastasis, tumor angiogenesis and reactive activation of host innate cells in the microenvironment at tumor periphery, thus, has highly practical application value.</p>