Effects Of Hypoxia On The Stemness Of Malignant Glioma Cells And Its Mechanism

Diffuse astrocytomas account for more than 60% of the primary brain neoplasms. Despite optimal clinical treatment, including aggressive surgical resection and advanced radiotherapy and chemotherapy, the prognosis for malignant astrocytomas remains poor: average survival time of patients with GBM after diagnosis is less than 1 year. Growing evidence in recent years has suggested that cancer is a stem cell disease. Cancer stem cells (CSCs), also termed as stem-like tumor cells, is a small subgroup of cells within tumor that prosses the ability of limitless selfrenewal, multipotency, as well as repopulating and maintaining a heterogeneous tumor. CSCs have important biological and therapeutic significance, which are believed to be the cell source of tumor initiation, progression and recurrence. Glioma is one of the earliest solid tumors which have been proved to contain CSCs. Our group has successfully isolated and cultured glioma stem cells (GSCs) from both of clinical freshly resected glioma specimens and widely used glioma cell lines, such as U87 and U251. GSCs, alike their possible normal counterpart, the neural stem cells, may need a specialized microenvironment, also named"niche", to regulate balanced self-renewal, differentiation and stem cell quiescence.Hypoxia, one of the basic biological features of malignant glioma, plays important roles in tumor progression through activating multiply signaling pathways that promote cell survival, motility and tumor angiogenesis. There is accumulating evidence that hypoxia might be a key element of GSCs niches, which contributes to promoting self-renewal capacity and maintaining undifferentiated state of GSCs in vitro. However, less evidence from clinical samples has been obtained to prove this novel idea. In addition, the molecular mechanism underlying the correlation between hypoxia and tumor stem cells is still remain unclear.Objectives1. To investigate the expression pattern of endogenous hypoxia marker CA IX in malignant glioma and to pay special attention to the correlation of stem cell marks expression with CA IX immunostaining in the malignant astrocytic tumors;2. To explore the clinicopathological significance of CA IX expression in malignant glioma. We also evaluate the application value of CA IX expression as a useful prognostic marker for glioma patients.3. To explore the effects of hypoxia on the cellular function of malignant glioma cells and to pay special attention to morphologic change, clone formation, expression of stem cell markers and differentiation.4. To investigate the effect of hypoxia on cellular function of glioma stem cells. Taking special care of the changes of cellular renewal, differentiation and expression of several stem cell markers.5. To study preliminarily the role of Notch signaling on maintaining stemness of GSCs induced by hypoxia.Materials and Methods1. Expression of hypoxia marker CA IX in malignant glioma: CA IX expression was detected by immunohistochemistry in 116 cases of astrocytomas. The association of CA IX with clinical parameters and patient's survival were analyzed. Coexpression of CA IX and CD133, Oct-4, Sox2, MMP-9 and CXCR4, as well as Ki-67 and p53, were detected by double- immunofluorescence and examined under a confocal microscope. Next, CA IX-positive and negative tumor cells were collected by laser capture microdissection and pooled for real-time quantitative PCR of mRNA of CA IX, CD133, Sox2, MMP9 and CXCR4.2. In vitro study of hypoxia on stemness regulation of malignant glioma cells: U87, U251 and 3 cases of primary cultured glioma cells were applied. Hypoxia culture model was established. Ultrastructural changes of tumor cells induced by hypoxia were observed by transmission electron microscopy. MTT assay was used to observe the effects of hypoxia on growth. Flow cytometry was applied to detect apoptosis and cell cycle. Transwell chamber was used for cell migration detection. Colony-forming ability of tumor cells was evaluated by colony formation assay. CD133 expression was detected by flow cytometry. Real-time quantitative PCR and western blot were applied to detect the mRNA or protein expression of stem cell markers and differentiation markers, respectively.3. In vitro study of hypoxia on stemness regulation of GSCs: Neurospheres were obtained from malignant glioma cells by conditioned medium sorting system and identified by expression of stem cell markers. Stem cell colony formation and differentiation assays were applied to observe the effects of hypoxia on the selfrenewal and differentiation of GSCs,respectively. CD133 expression was detected by flow cytometry. Expression of stem cell markers and differentiation marker were detected by immunofluorescence staining and western blot.4. Preliminary study of the effect of Notch signaling on the stemness regulation of GSCs induced by hypoxia:Western blot was used to detect the protein level of several key markers of Notch signaling. Appropriate concentration (5μg/ml) of DAPT, a specific inhibitor of Notch signaling, was determined. Stem cell colony formation and differentiation assays were applied to evaluate the effect of hypoxia on the selfrenewal and differentiation of GSCs, respectively.Results1. CA IX expression in the malignant gliomas and its clinicopathological significance:CA IX was overexpressed in malignant gliomas, and mostly localize in the peri-necrotic regions in malignant glioma and the invasive front of low-grade gliomas. CA IX expression correlated with histologic grade and tumor size. Survival data analysis showed that the overall survival and progression-free survival of CA IX(++) group were significantly lower than those of CA IX(+) or CA IX(-)group. Multivariate analysis showed that CA IX (++) expression in malignant glioma was an independent negative prognostic marker.2. Relationship between CA IX,stem cell markers and invasive markers in the malignant glioma tissues: Double-labeled immunofluorescence showed that the number of CD133, Oct4, Sox-2 or CXCR4 positive tumor cells in CA IX(++) regions was significantly higher than that of in CA IX(-)regions. While no correlation was found between CA IX expression and p53 or Ki-67 immunostaining. mRNA levels of CA IX, Oct4, Sox2, and CXCR4 in microdissected CA IX (+) cells were significantly higher than those in the corresponding CA IX-negative tumor cells.3. In vitro study of hypoxia on the stemness regulation of malignant glioma cells: In hypoxia condition, obvious morphologic changes occurred. Generally, the tumor cells changed from mature to "childish", showed as organelles decreased, glial filaments disappeared and nucleolar appeared. Hypoxia significantly enhanced colony formation ability of tumor cells and increased the proportion of compact colony formation. Hypoxia enhanced tumor cell migration. Flow cytometry analysis showed that hypoxia significantly increased CD133-positive cell ratio. Under hypoxia condition, mRNA and protein expression of stem cell marker increased, while GFAP expression was significantly decreased.4. In vitro study of hypoxia on the stemness regulation of GSCs: Neurospheres were obtained from malignant glioma cell by conditioned medium sorting system and identified by expression of stem cell markers (CD133, Oct-4 and Nestin). Under hypoxia condition, the number and size of the spheres significantly increased, as compared with those in control group, while the differentiation process was inhibited .Upon hypoxia, the ratio of CD133 positive cells increased significantly. The Oct-4 and Sox-2 protein level were also upregulated.5. Effect of Notch signaling on stemness regulation of GSCs induced by hypoxia: Upon hypoxia treatment, protein of several key molecules of Notch signaling, including Notch1, Notch1 ICD and Hes-1, were upregulated. DAPT inhibited Notch1 protein expression in a dose-dependent manner. 5ug/ml DAPT could reach nearly 80% of inhibition. 5ug/ml DAPT could obviously inhibit the selfrenewal process induced by hypoxia.Conclusion1. Endogeneous hypoxia marker CA IX is overexpressed in malignant glioma, which implies higher histological grade and poorer prognosis. Tumor progression mediated by CA IX may be associated with higher histologic grade and poorer outcome.2. We identified that more tumor cells with stem cell phenotype existed in CA IX positive region. mRNA expression of stem cell markers in CA IX positive regions were significantly higher than those in CA IX negative regions. These results provided strong direct evidence based on clinical samples to support the novel idea of"hypoxia as TSCs niches".3. Hypoxia can induce differentiated tumor cells into undifferentiated state, thereby regaining some features of stem cells.4. Hypoxia can maintain or increase the stemness of GSCs, as well as inhibit differentiation.5. Notch signaling might play an essential role in the stemness maintain or increase of GSCs induced by hypoxia.