| Background:Exploration of tumor stem cells impelled us to recognize the biological behaviors of malignances from a new perspective. Tumor stem cells are identified to be the root of tumorigenesis, invasion, angiogenesis and treatment resistance. It is also regarded as the important target to treat tumor. Chronic inflammation often accompany with formation, progression, invasion and metastasis of tumor. Inflammation cells and inflammation medium are crucial elements of tumor microenvironment. Chronic inflammation can induce tumorigenesis. Malignances can also induce tumor related inflammation which promotes development and progression of tumor and plays key roles in regulation of malignant biological behaviors. Recent researches suggested CXCL8 and their receptor, CXCL1, could promote the“stemness”maintenance of breast cancer stem cells, Foxp3+IL-17+ T cells could promote development of cancer-initiating cells in colorectal cancer, Glioblastoma cancer-initiating cells inhibit T-cell proliferation and effector responses by the signal transducers and activators of transcription 3 pathway.These results indicated the interaction between tumor stem cells and inflammation microenvironment regulates tumor malignant behaviors. Investigation on the interaction between TSCs and tumor related inflammation may provide new thoughts and experimental evidences for treatment strategy on targeting TSCsGSCs have been confirmed in malignant glioma and the relationship between GSCs and glioma related inflammation need further exploration. Due to the particularity of glioma growth areas, there are large differences in inflammation microenvironment of glioma. Microglia/macrophages are the most important immune infiltrating cells. Because there is no biomarker which can distinguish macrophage from microglia, thus, both are often regarded as a whole, glioma associated microglia/macrophages (GAM/Ms). Recent investigations indicated, GAM/Ms distributed within malignant glioma and para-glioma tissues. The amount of GAM/Ms surpass the total amount of other kinds of immune cells. Glioma derived growth factors and cytokines induce GAM/Ms infiltration. However, GAM/Ms exhibit immunosuppressive phenotype, promote the growth and invasion of glioma cells, boost tumor angiogenesis and immune evasion of tumor cells. Whether there is interaction between GSCs and infiltration of GAM/Ms which exhibit immunosuppressive phenotype? Whether the interaction between GSCs and GAM/Ms further promotes the growth and progress of glioma? These questions need further investigation.GSCs are the roots of tumorigenesis and recurrence of glioma, stem cell niche is the basic element for survival of stem cells and“stemness”maintenance. But the architecture of GSC-niche is unclear. Recent research indicated GSC-niche is a peri-vascular niche, micro vessels and endothelial cells provide growth factors and activated signals for GSCs survival and“stemness”maintenance. Accumulating evidences have also confirmed that inflammation medium and inflammation cells promote“stemness”maintenance and invasion of GSCs. However, whether inflammation microenvironment is also the constituent of GSC-niche? Inflammation is defined as the blood vessel-centered pathological process. Considered the proven peri-vascular niche of GSCs, we are necessary to explore the interaction between GSCs and GAM/Ms. Research results may lay the foundation for further elaboration the architecture characteristics of GSC-niche.Materials and methods:1. To isolate and culture GL261 neurosphere (GL261-NS) from mouse malignant glioma cell line GL261 using conditioned medium sorting system.2. To test the expression of the stem cell markers in GL261-NS and the differentiated markers in GL261 adhesive cells (GL261-AC) using immunofluorescence and flow cytometry.3. To detect the self-renewal ability of GL261-NS and GL261-AC using limited dilution assay and clone formation assay.4. Syngeneic intracranial transplant glioma model was established by injecting GL261-NS into caudate nucleus of C57BL/6 mice through stereotaxie apparatus. 5. Immune cells infiltration in primary malignant glioma, intracranial transplant glioma of GSCs in SCID mice and in C57BL/6 mice were detected.6. The relationship between GSCs density and GAM/Ms infiltration was observed in 33 cases of primary astrocytoma with different WHO grades by immunohistochemistry.7. To isolate and culture primary GSCs from human malignant glioma using conditioned medium sorting system. Primary adhesive glioma cells (AGCs) were obtained by inducing differentiation of GSCs.8. Transcriptional levels of several kinds of cytokines which can induce macrophage recruitment were detected by Realtime-PCR.9. The abilities of GSCs and AGCs to induce primary microglia recruitment were detected by Transwell chamber assay and antibody blocking assay.10. The impact of GSCs on GAM/Ms infiltration was observed by syngeneic intracranial transplant glioma model11. The expression levels of VEGF-A, M-CSF and TGF-β1 were tested by ELISA.12. The inhibitory effects of microglia’s phagocytosis induced by GL261-NS and GL261-AC were observed by phagocytosis assay.13. The proliferation effects of T cells induced by microglia which were pretreated by GL261-NS and GL261-AC were observed by Transwell co-culture system and CCK8 kit.Results:1. Isolation and culture of GSCs from glioma cell line GL261.(1) Neurosphere could be obtained from mouse malignant glioma cell line GL261 by conditioned medium sorting system. GSCs were enriched in GL261-NS, and expressed stem cell markers CD133, Nestin and Olig2.(2) GL261-NS could differentiate in culture medium with fetal calf serum and became GL261-AC which partially expressed differentiated markers GAFP, MAP2 and MBP.(3) GL261-NS possessed stronger self-renewal capacity than GL261-AC. 57.31%±8.53% GL261-NS could self-renew, but only 2.62%±0.23% GL261-AC could self-renew.(4) Intracranial transplant tumor assay confirmed that GL261-NS possessed stronger tumor-forming ability than GL261-AC. Mice bearing GL261-NS had longer survival period than mice bearing GL261-AC. 2. Establishment of syngeneic intracranial transplant glioma model Compared with intracranial transplant glioma model in SCID mice, the syngeneic intracranial transplant glioma model could better simulate immune microenvironment of primary malignant glioma and assessed the biological behaviors of GSCs more accurately3. GSCs played a predominant role in inducing GAM/Ms infiltration(1) GAM/Ms infiltration varied largely in astrocytomas with different histological grades. The higher the histological grade of astrocytoma, the greater the number of infiltrating GAM/Ms in tumor tissue. The density of GSCs positively correlated with the histological grade of malignancies and there was a positive correlation between the infiltration of GAM/Ms and the density of GSCs.(2) GSCs were arranged in nests or cords and, intriguingly, the majority of GAM/Ms were located around the hotspots of GSCs.(3) Primary GSCs produced more CCL2, CCL5, CCL7, VEGF-A and NTS than do the AGCs.(4) Primary GSCs possessed stronger capacity of microglia recruitment and the improved capacity could be ruined by preincubation with neutralizing antibodies in vitro.(5) The syngeneic intracranial transplant glioma model confirmed that GAM/M infiltration in GL261-NS tumors were much more obvious than in GL261-AC tumors.4. GSCs induced immunosuppressive phenotype of GAM/Ms(1) GL261-NS which enriched GSCs expressed kinds of cytokine which induced immunosuppressive GAM/Ms. Compared with GL261-AC, GL261-NS expressed more VEGF-A and M-CSF and had stronger capacity to induce immunosuppressive microenvironment.(2) Microglia BV2 pretreated with GL261-NS expressed more p-STAT3 than microglia BV2 pretreated with GL261-AC and control medium.(3) Microglia BV2 pretreated by GL261-NS and GL261-AC could both inhibit proliferation of T cells. But the inhibitory effects of GL261-NS treatment group and GL261-AC treatment group had no significant difference.(4) GL261-NS and GL261-AC could inhibit the phagocytosis of BV2 effectively. Compared with GL261-AC, GL261-NS had stronger capacity to inhibit BV2 phagocytosis.Conclusion: 1. GSCs are enriched in GL261-NS, GL261-NS have self-renewal capacity and multiple differentiative potent. GL261-NS possess stronger tumor-forming ability than GL261-AC.2. The syngeneic GSCs intracranial transplant glioma model is established by injecting GL261-NS into caudate nucleus of C57BL/6 mice. Compared with intracranial transplant glioma model in SCID mice, the syngeneic GSCs intracranial transplant glioma model could better simulate immune microenvironment of primary malignant glioma and assessed the biological behaviors of GSCs more accurately3. GSCs play a predominant role in inducing GAM/Ms infiltration.4. GSCs induce immunosuppressive phenotype of GAM/Ms. |
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