HIF1α regulates glioma chemosensitivity through the transformation between differentiation and dedifferentiation in various oxygen levels
Chemotherapy plays a significant role in glioma treatment; however, it has limited effectiveness in extending the life expectancies of glioma patients. Traditional studies have attributed this lack of efficacy to glioma stem cells (GSCs) and their high resistance to chemotherapy, and hypoxia worsens this issue. In contrast, hyperoxia effectively alleviates hypoxia in glioma and sensitizes glioma cells to chemotherapy. In a summary of traditional studies, the majority of researchers overlooked the influence of hypoxia on differentiated cells because they only focused on the maintenance of GSCs stemness, which thus resulted in chemoresistance. Because of this background, we hypothesized that GSCs may be induced through dedifferentiation under hypoxic conditions, and hypoxia maintains GSCs stemness, which thus leads to resistance to chemotherapy. In contrast, hyperoxia inhibits the dedifferentiation process and promotes GSCs differentiation, which increases the sensitization of glioma cells to chemotherapy. Hypoxia-inducible factor-1α (HIF1α) contributes substantially to the stemness maintenance of GSCs and resistance of glioma to chemotherapy; thus, we investigated whether HIF1α regulates the resistance or sensitization of glioma cells to chemotherapy in different oxygen levels. It highlights a novel viewpoint on glioma chemosensitivity from the transformation between dedifferentiation and differentiation in different oxygen levels.CD133+CD15+NESTIN+ GSCs sorted from GL261 and U87 cells were cultured in stem cell medium (DMEM/F12 + EGF + FGF2 + B27), and the cells grew as a suspension with a sphere morphology (Fig. 1A). Immunofluorescence indicated these neurospheres highly expressed stem cell markers CD133, CD15 and NESTIN and the chemoresistance-related proteins ABCG2 and MGMT (Fig. 1B,C). Furthermore, western blot and RT-qPCR assays demonstrated an absolute increase in CD133, CD15, NESTIN, ABCG2 and MGMT expression in GSCs compared with CD133−CD15−NESTIN− cells (Fig. 1D,E, Supplementary Figure S8A,B). We subsequently determined that the GSCs were arrested in G0/G1 (Fig. 1F), and fewer of these cells underwent apoptosis after TMZ (100 μM) exposure compared with CD133−CD15−NESTIN− cells exposed to the same treatments (Fig. 1G).GL261 and U87 cells were considered glioma cell lines. We used magnetic cell sorting (MACS; Miltenyi Biotech, Bergisch-Gladbach, Germany) to sort differentiated CD133−CD15−NESTIN− glioma cells and CD133+CD15+NESTIN+ glioma stem cells45 and the detailed methods of MACS were shown in supplementary materials. GL261 and U87 cells were initially incubated in DMEM/F12 + 10%FBS medium at 21%O2 to maintain cell growth. The glioma cells were subsequently digested by 0.25% trypsin, centrifuged and suspended with DMEM/F12 + 10%FBS medium. Second, the cells were centrifuged again and re-suspended in PBS with 0.08%EDTA and 0.5%BSA (PBSE; 108 cells/500 μl). Polyclonal rabbit anti-mouse or human CD133+ IgGs (Miltenyi Biotech, Germany) was added and maintained at 4 °C for 15 min. The cells were subsequently washed with PBS that contained 1%BSA and centrifuged again. The cells were suspended in PBSE (108 cells/300 μl), and goat anti-rabbit IgG MicroBeads (Miltenyi Biotech, Germany) was added to the cell suspension and cultured at 10 °C. After 15 min, the cells were washed at least twice with PBSE. The cell number was counted, and the cells were suspended in 500 μl of PBSE. The cell suspension was poured into a column reservoir, which was placed by a miniMACS magnet and flushed with 500 μl of PBSE; unlabeled nonmagnetic CD133− cells were collected in a culture flask. The cells retained on the magnet were CD133+ cells. We repeated these steps at least three times to purify the sorted cells. We subsequently used the same methods to sort CD15− and NESTIN− cells from sorted CD133− cells. CD133+CD15+NESTIN+ glioma stem cells were also sorted as described. Sorted CD133−CD15−NESTIN− glioma cells were cultured in DMEM/F12 + 10%FBS culture medium, and CD133+CD15+NESTIN+ glioma stem cells were cultured in stem cell medium (DMEM/F12 + EGF + FGF2 + B27).This work was supported by the National Natural Science Foundation of China (NSFC 81272784; 81672493), the Science Foundation of Southwest Hospital (SWH2015QN10) and the Key laboratory of tumor immunology of Ministry of Education (2015sjz114).The authors declare that they have no competing interests.Pan Wang and Wenwu Wan contributed equally to this work.Electronic supplementary materialSupplementary information accompanies this paper at doi:10.1038/s41598-017-06086-2Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.