Dedifferentiation Of Glioma Cells Induced By Hypoxia: An In Vivo Study

Background and Objective:In the central nervous system, with the highest degree of malignancy and mortality tumor is glioma. Although there are standard treatments of surgical resection, radiotherapy and chemotherapy, the prognosis is poor.Tumor recurrence after surgery is easily.The recurrent tumor has a higher degree of malignancy, more tolerable to chemoradiotherapy. Therefore, we need to develop new treatments to improve the prognosis of gliom a patients. Hypoxia is an important feature of solid tumors, the presence of tumor stem cells has also been confirmed, and tumor stem cells present in a hypoxic microenvironment. Hypoxia contribute to the occurrence, development and chemoradiotherapy tolerance of glioma via promote self-renewal of glioma stem cells. However, there is little reports about whether glioma cells involve in the malignant progression of glioma and regulated by hypoxia microenvironment at present. Therefore, we intend to demonstrate that hypoxia can induce dedifferentiation of glioma cell into glioma stem-like cells, further aggravate the malignant progression of glioma in vivo.Methods:Cultured GL261 cells, and isolated CD133- GL261 cells by immunomagnetic sorting. Some cells were cultured in the hypoxia(1% O2) 21 days and the others in normoxia(21% O2) 21 days. Infected the CD133- GL261 cells by luc lentivirus to gain stably transfected GL261-Luc cells, and then divided C57 mice into 4 groups randomly. Group 1: mice were intracerebrally injected by GL261-Luc cells which were cultured in the hypoxia(1% O2) 21 days(1*104 per mouse); group 2: mice were intracerebrally injected by GL261-Luc cells which were cultured in normoxia(21% O2) 21 days. Mice in these 2 groups were fed in normoxia(21%O2,5%CO2,74%N2). We used some of them to detect the tumor growth through bioluminescence of cancer cells at day 5, 10, 15, 20 and 25, respectively, and the survival rate was examined in 30 days by the rest of the mice. HE staining was used to detect whether tumor had been formed. Mice in group 3 and group 4 were intracerebrally injected by GL261-Luc cells which were cultured in the normoxia(21% O2) 21days(5*104 per mouse). Mice in group 3 were feed in low oxygen environment(10%O2,5%CO2,85%N2) and group 4 in normoxia(21%O2,5%CO2,74%N2). The tumor growth and survival rate were detected as the same way of group 1 and group 2. Im munohistochemical staining, western blot and RT-PCR were used to demonstrate the expression of tumor stem cell markers SOX-2, OCT-4, KLF-4, Nanog, CD133.Results:1、Living imaging between group 1 and group 2: mice in group 1 had significant tumorigenesis in 10 days and tumor volume increased obviously after 25 days. Mice in group 2 had no significant tumorigenesis. T test *P<0.001.2、Living imaging between group 3 and group 4: with the time extended, tumor volume in group 3 increased but group 4 had no significant tumorigenesis. T test *P<0.001.3、Survival time analysis: mice in group 1 had a lower survival rate than that in group 2 and mice in group 3 had a lower survival rate than that in group 4 in 30 days,*P<0.0001.4、Mice in group 1 and group 3 had significant tumorigenesis and mice in group 2 and group 4 had no significant tumorigenesis. These results were confirmed by HE staining.5、Tumer stem cell markers detection:(1) Immunohistochemical staining showed SOX-2, OCT-4, KLF-4, Nanog, CD133 were highly expressed in tumors in group 3.(2) The expression of RNAs of SOX-2, OCT-4, KLF-4, Nanog and CD133 in group 3 were higher than the other two groups through RT-PCR. One-way ANOVA *P≤0.001, while the comparison between normal C57 mice and group 4 was *P>0.05.(3) Western blot showed compared with normal C57 mice and mice in group 4, the proteins of SOX-2, OCT-4, KLF-4, Nanog, CD133 had a higher expression in mice of group 3. One-way ANOVA *P<0.05. While no significant difference was found between the normal C57 mice and group 4.Conclusion:1、After injecting CD133-GL261 glioma cells cultured in hypoxia(1% O2) into C57 mice brain, the mice had tumorigenesis and a lower survival rate than that in control group. These phenomemon suggest that hypoxia can induce the formation of glioma stem-like cells in vitro.2、After injecting CD133-GL261 glioma cells cultured in normoxia(21% O2) into C57 mice brain, the one fed in hypoxia(10% O2) had high tumorigenesis and the expression of tumor stem cell markers SOX-2, OCT-4, KLF-4, Nanog, CD133 in these mice were positive. Besides, mice fed in hypoxia(10% O2) had a significant lower survival rate compared with control group. There results demonstrate that hypoxia in vivo can induce the forming of glioma stem-like cells.3、Above all, the hypoxic microenvironment in glioma can induce glioma cells dedifferentiate into glioma stem-like cells, further aggravate the malignant progression of glioma.