Effect Mechanisum Of Brain Tumor Stem Cells On The Treg Cells And Th17Cells

Background and ObjectionGlioma is the most common primary tumor in the central nervous system (CNS), which is high mortality and a serious threat to human health. The main methods of treatment for glioma are surgery, radiotherapy and chemotherapy. However, the overall outcome is still unsatisfactory. The survival rate is still low. According to statistics, malignant brain tumors accounted for2%of all human cancer patients, accounting for all the riskof children tumor as20～25%. The peak age of onset was fromed30to40years old, or10to20years old. Especially, the morbidity of glioma was the highest among the brain tumors, accounting about40.49%in all the malignant brain tumors. The median survival time of glioblastoma, after diagnosis, gegerally was15months. Due to the un-completed differentiation and infiltration to deep nerve tissue, the glioma were difficult to be effectively eradicated by the conventional antitumor measures including surgery, radiotherapy and chemotherapy, which was the main reason of its higher recurrence rate. All of these described above were closed to the biological characteristics of glioma. Glioma is infliltrating growth, and was hard to be found the boundaries between normal brain tissue and tumor. Therefore, the simple surgery was difficult to remove total tumor. On the other hands, the traditional chemotherapy effect was still very limited because of the existence of the blood brain barrier (BBB) and the high resistance of glioma to toxicity drugs. Microsurgery technology has made a lot of progress, and the safetyscope of the operation had been expanded, mean while the related complications was significantly reduced. The applications including routine radiotherapy and chemotherapy with oral temozolomide after surgery for the glioma individual, in certain degree, had improved the patient survival. However, the technological advancement did not improve the survival time of patients with malignant glioma, in which the biggest difficulties still suffered from the recurrence. In view of this, the effective treatment and control recurrence of malignant glioma have been being as the key and difficult research point in the neurosurgery field. It is much meaningful to study and explore the new ways to treat glioma and inhibit recurrence.It was well known that there were rarely cells in glioma, which showed the characteristics of stem cells defined as brain tumor stem cells (BTSCs). Ignatova et al first reported that the BTSCs existed in the malignant glioma. They cultured glioma cells under neural stem cells (NSCs) culture condition, and found that the sphere formed, which expressed NSCs markers (Nestin). The differentiated cells also expressed the neurons specific enolase enzyme (NSE), glial cells fiber acid protein (GFAP) and neurons betaâ…¢microtubules protein (neuronal (3-III tubulin) respectively. Therefore, the results showed that this kind of cells possessed the NSCs characteristics and were able to differentiate into neurons and glial-like cells. They called them as tumor stem-like cells, and believed that cells were important to the glioma genesis. Since then, Singh et al found that nearly all of the brain tumor cells were just proliferated from a small part of the cells expressing positive CD133. They called them as brain tumor stem cells (BTSCs), which could propagate and differentiate into neurons and (or) glial cells of glioma, and the phenotype and proportion showed the same with the primary glioma. These BTSCs also could formthe new tumor cells in culture, by which it could be confirmed that they were just the tumor stem cells but not the polluted normal NSCs. Hemmati et al (2003) also found that the minority of glioma cells in the children medulloblastoma and glioma possessed the similar properties of NSCs. The differences beween the BTSCs and the NSCs just presented that the BTSCs could differentiated into the tumor cells, which showed the phenotype and karyotypic as same as the primary glioma.The common features of BTSCs include as bellow:(1)The positive expression of NSCs specific markers, such as Nestin, CD133, etc.(2)To possess a self-renewal and proliferation ability, and to become eternal functional cells.(3)To be able to differentiate the similar phenotypes and the karyotype as same as the original tumors following the transplantation.CD25+CD4+regulatory T cells (Treg cells) were found in recent years. They belong to the T cells subsets and play a negative function in immune response through the cell-cell contact and the inhibited cytokines.Treg cells were first reported by Sakaguchi in1995. Treg cells showed two functions including the low reactive and the immune suppression, which play the important roles in maintenacing the immune steady and help the tumor cells escape the immune response. Treg cells inhibited T cells function, and also reduced the body’s immune response to the tumor cells at the same time, which indirectly promoted the growth of tumor cells. In a variety of malignant tumor tissue (such as breast cancer, ovarian cancer, lung cancer, liver cancer and malignant lymphoma, etc), there were a great quantity of Treg cells infiltrate, and the lymphocyte proportion of the Treg cells in the peripheral blood of patients with tumor also increased significantly. And, there was a negative correlation between the number of Treg cells and the development of cancer. It will be helpful for enhancing antitumor ability and recovering the immune function, as soon as to reduce the number of Treg cells or to inhibit its function. Forked head/wings shape helix transcription factors (FOXP3), which specifically expressed in Treg cells, is the key genes to control development and function of Treg cells.Th17cells were as the new members of the T help cells, which were originally found in autoimmune diseases, and expressed positive CD4and IL-17. The naive CD4+T cells were influenced by TGF-beta and IL-6cytokines to differentiated into Th17cells, depending on transcription factors RORγt.Otherwise, the cytokinss such as IL-23and IL-1β were also thought as the important parts for the differentiation and function of Th17cells. There were many studies of Th17function on the Th17cells in infection and autoimmune diseases.It was known that the Thl7cells participated in the pathologic process of infection and inflammation mainly through the positive expression of IL-17. At the same time, the function of Th17cells in tumor also were closely payed attentions to the research field. The main effect cytokine of Th17cells, IL-17, was confirmed at least through the following several ways to promote the cancer development.(1).Raising the positive expression of VEGF, CD31and other factors, so as to promote the growth of blood vessels, accelerate the tumor growth.(2).Upregulating the signal ways of IL-6and STAT3, to promote cancer progression.(3).Down regulating the IL-12Rβ molecular expression on the Thl cells surface, to reduce the Thl cells function and to resist the effective tumor immunity.(4).Making the CD8+cells to express IL-17positively, and to lose their cytotoxic effects, to resist the cells apoptosis.Upon the views described above, it was well understood that the BTSCs were closely related to the development of glioma. Both Treg cells and Th17cells promoted this process by some of mechanisms such as immunology and inflammation. Then, what were the relationships between Treg cells and Th17cells, or between the BTSCs and Treg Cells, or between BTSCs and Thl7cells? Whether or not the BTSCs resulted in the immunological tolerance of glioma by Treg cells or Th17cells?It was well known that there was an opposition state on the function and differentiation, between the Treg cells mediating immunologic tolerance and the Th17cells mediating inflammatory reaction. In normal state, there was a balance between these two of cells. As soon as there was an abnormal in the body, the balance between Treg cells and Th17would be broken. Some of researches showed that the IL-6, a kind of cytokine secreted by the active dentritic cells, was a key factor for priming the CD4+cells and making Treg cells or Th17cells to differentiate. On the other hand, RORyt, a member of hormone receptors superfamily, is the main transcription factor during Th17cells differentiation.Foxp3, as the important signature molecular of the Treg cells, can combine with the RORyt, to reduce the inhibition of RORyt to IL-17expression.It was known that the number of Th17cells was negatively related to the number of Treg cells in a same tumor, which implied there was the interaction between the two kinds of cells. Some researches showed the mature Treg cells aound the peripheral nerver of mouse might transfer into the Th17cells, which easily appeared in the process of infection or IL-6production. Otherwise, high level expression of CD30in Treg cells, which closely related to tumor and belonged to an outer-core nucleotidase with the functions transforming the ATP to the adenosine, might inhibit the Th17cells activity by blocking the adenosine pathway. Therefore, the prohibition of Th17cells development would escape the defense reaction of immunological system, because of the possible immune defence mediated by Th17cells.Although there was rare the research about relationship among glioma, Treg cells and Th17cells, it was recently reported that the Th17and Treg cells might increase along with the time-lapse, and that the polarization of Th17cells might accomplished by the interaction of cytokines derived from the Treg cells and glioma microenviroment. It implied that no mater Thl7cells or Treg cells in the glioma tissue played an important immune role.Considering the original position of BTSCs in glioma development and the interaction between Treg cells and Th17cells, we initialy explored the relationship among BTSCs, Treg cells and Th17cells in the same individuals. By studing synchronously, we expected to clear the effect position of BTSCs in the immune environment related to glioma.Methods and materialsThere are three parts of experiment in this research.Part1Isolation, purification and characteristics of BTSCsTo get the glioma tissues from patients during surgery. After digested and dissociated, primary glioma cells were cultured in the DMEM/F12medium containing10%FBS for3～5days. Then, parts of primary cells were examined with Flow Cytometry to detect the rate of CD133+cells in the primary cells. We cultured human primary glioma cells and rat glioma cell lines (9L), through the serum-free culture method [DMEM/F12medium with B27, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF)]. After10～20days culture, the suspending tumor cell spheres (BTSCs sphreres) would be observed. We used flow cytometry and immunocytochemistry method was used to test the positive expressions of CD133and Nestin antibody on these BTSCs spheres. We also used CCK-8method to test the survival condition of these cells. Through the stereotactic, we transplanted rat glioma cells lines into F344or Wistar rats, in order to observe rat survival time and tumor growthafter transplantation. Part2:BTSCs, Treg cells and the related cytokinesAfter glioma animal models finished in the first part, we used flow cytometry to test the content of Treg cells in peripheral blood, upon the expressing rate of CD4å'ŒCD4+FOXP3+cells, and also used the enzyme-linked immuno sorbent assay (ELISA) toexamine cytokines such as IL-10, IL-6, TGF-β, and IFN-y, etc in the peripheral blood.On the17th day after transplantation, the glioma modeled rats were sacrificed and prepared biopsy. The immunohistochemical assays for the expression of Nestin, CD133and Foxp3protein were performed. The quantitative PCR to detect the expressions of Nestin, CD133and Foxp3gene were also employed in this study. After collecting glioma specimens and peripheral blood from a same individual, we used the flow cytometry to test the CD4+Foxp3+cells (Treg cells) and the quantitative PCR to detect the gene expression of Nestin, CD133and Foxp3in tumor or in peripheral blood.Part3:BTSCs, Th17cells and the related cytokinesAfter glioma animal models finished in the first part, we used the flow cytometry which was employed to test the positive percentage of CD4+IL17+cells in CD4+cells in peripheral blood, and also the enzyme-linked immuno sorbent assay (ELISA) was used to examine the cytokines such as IL-17, IL-23, etc in the peripheral blood. On the17th day after transplantation, the glioma modeled rats were sacrificed and prepared biopsy. We used immunohistochemical technique to assay the expression of Nestin, CD133and IL-17protein in glioma tissue. We used the quantitative PCR to detect the expressions of Nestin, CD133and IL-17genes. After collecting the glioma specimens and peripheral blood from the individuls, we used flow cytometry to test the Treg cells percentage in peripheral blood upon the positive expressing of CD4+IL17+in CD4+cell, and quantitative PCR to detect the gene expressions of Nestin, CD133and IL-17in tumor. Statistical analysisAll data were analyzed through statistical software SPSS13.0. Results are expressed as mean±SD. The t test was used for comparisons of two groups, and the one way ANOVA was used for comparisons of multiple groups. Kaplan-meier was used for survival analysis. Difference is considered as the significant at P<0.05.Results1. BTSCs and their characteristicWe successfully separated and cultured tumor spheres (BTSCs spheres) from human primary gliomas and rat9L glioma cell lines. The result of immunohistochemical assay approved spheres expressed CD133and Nestin protein molecules positively. After cultured in normal serum medium, these tumor spheres (BTSCs spheres) became adherent cells, which positively expressd GFAP or NSE protein (see chart1-1,1-2,1-3). After the general gliocytes or the tumor spheres-formed gliocytes were transplanted into the rat brain separatelly, the rats with the spheres-formed gliocytes showed the less survival time (22.00±0.557days), comparing to the ones with the general adherent glioma cells (27.00±2.309days)(Fig.1-5). We also found that the onset of neurological symptoms like seizures, hemiplegia in rats transplanted with gegeral adherent gliocytes was much later, compared with the ones with tumor sphere-formed gliocytes. Rats planted with glioma cell-line subcutaneously, were sacrificed on the21st day after planting. We measured the size of tumor tissues. The statistical analysis showed that the size of tumor tissue which planted with tumor spheres was much larger than the one planted with general gliocytes. After analyzing the human primary glioma and survival time of patients, we found that the survival period of patients whose glioma could be cultured into tumor spheres in vitro, was much shorter, comparing with the ones whose glioma could not be cultured into tumor spheres in vitro (Tab.1-2,1-3). 2. BTSCs, and Treg cellsFor animal specimens:ELISA test showed that the quantity of IL-10and TGF-β peripheral blood of the rat model was significantly different, comparing with the healthy controls (Fig2-2,2-3). At the same time, the positive TGFβ-express inperipheral blood showed the significant difference between the modeled rats with tumor sphere-formed gliocytes and the ones with general adherent gliocytes. Flow cytometry test results indicated that the number of CD4+Foxp3+cells was significantly different between the normal rats (6.24±1.832%) and the in situ glioma (9.82±2.663%). There was also significantly different about the CD4+Foxp3+number in peripheral blood, between the modeled rats with tumor sphere-formed gliocytes (11.52±2.663%) and the ones with general adherent gliocytes (8.12±2.493%)(Fig.2-1). Quantitative PCR and immunohistochemical detection also confirmed the existence of CD4+Foxp3+cells (Fig.2-4,2-6)For human glioma specimen:The flow cytometry test showed that the number of CD4+Foxp3+cells in peripheral blood were significantly different between normal individuals and glioma-loaded ones (4.640±0.817%vs.8.713±2.178%)(Fig.2-8). Immunohistochemical approved that CD4+Foxp3+cells existed in the glioma tissues. Quantitative PCR detection also displayed the positive expressions of CD133, Nestin and Foxp3increased in glioma tissues along with thepathological level increasing (Fig.2-9,2-10)3. BTSCs, Th17cells and the related cytokinesFor animal specimens:ELISA test showed the quantity of IL-17and IL-23in peripheral blood of the modeled rat was not significantly different with healthy controls (Fig.3-2,3-3). At the same time, the positive expressing of IL-17and IL-23difference between the modeled rats with tumor sphere-formed gliocytes and the ones with general adherent gliocytes (Fig.3-2,3-3). Flow cytometry test results indicated that the number of CD4+IL-17+cells was not significant difference between the normal rats and in situ glioma rat model (Fig.3-1). There was no significant difference between the tumor sphere-graftedanimal models and the general adherent cells-grafted ones on the number of CD4+IL-17+cells in peripheral blood (Fig.3-4) Quantitative PCR and immunohistochemical detection also proved the existence of CD4+IL-17+cells.For human glioma specimen:The flow cytometry test showed that the number of CD4+IL-17+cells in peripheral blood was not significantly different between normal individuals and the glioma-loaded ones (Fig.2-8). Immunohistochemical approved that CD4+IL-17+cells existed in the glioma tissues. Quantitative PCR detection displayed the positive expression of IL-17. There was no significant difference about CD4+IL-17+number or positive IL-7expression between the two of modeled rats (Fig.2-9,2-10).Conclusion1. Comparing the individuals with the general gliocytes but tumor spheres-free, the ones with the positive tumor spheres showed the much shorter of life span.2. Comparing the individuals with the general gliocytes but tumor spheres-free, the ones with the positive tumor spheres showed the much more number of Treg cells in peripheral blood, and much higher positive expression of TGF-β, along with the BTSCs increasing.3. Comparing the individuals with the general gliocytes but tumor spheres-free, the ones with the positive tumor spheres did not show the increased number of Th17cells. There was no significant difference about the positive expressions of IL-17or IL-23between the two of modeled rats.