HIF1α regulates single differentiated glioma cell dedifferentiation to stem-like cell phenotypes with high tumorigenic potential under hypoxia
The standard treatment for Glioblastoma multiforme (GBM) is surgical resection and subsequent radiotherapy and chemotherapy. Surgical resection of GBM is typically restricted because of its invasive growth, which results in residual tumor cells including glioma stem cells (GSCs) and differentiated cells. Recurrence has been previously thought to occur as a result of these GSCs, and hypoxic microenvironment maintains the GSCs stemness also plays an important role. Summarizing traditional studies and we find many researchers ignored the influence of hypoxia on differentiated cells. We hypothesized that the residual differentiated cells may be dedifferentiated to GSC-like cells under hypoxia and play a crucial role in the rapid, high-frequency recurrence of GBM. Therefore, isolated CD133–CD15–NESTIN– cells were prepared as single-cell culture and treated with hypoxia. More than 95% of the surviving single differentiated CD133–CD15–NESTIN– cell dedifferentiated into tumorigenic CD133+CD15+NESTIN+ GSCs, and this process was regulated by hypoxia inducible factor-1α. Moreover, the serum also played an important role in this dedifferentiation. These findings challenge the traditional glioma cell heterogeneity model, cell division model and glioma malignancy development model. Our study also highlights the mechanism of GBM recurrence and the importance of anti-hypoxia therapy. In addition to GSCs, residual differentiated tumor cells also substantially contribute to treatment resistance and the rapid, high recurrence of GBM.We first investigated whether hypoxia induced single differentiated CD133−CD15−NESTIN− glioma cell into neurosphere. Under hypoxia more than 60% cells survived at 3 days (d). Most surviving cells (95.38% ± 5.83 from GL261 and 99.10% ± 0.78 from U87) formed neurospheres at 21 d. However, under normoxia only 5.05% ± 1.12 from GL261 and 1.31% ± 1.32 from U87 cells formed sparse, irregular and non-adherent aggregates. Most surviving cells remained a single cell and died at 21 d (Figure 1B–1C, Supplementary Figure 1 and Supplementary Table 1). Besides, the results of Trypan blue showed almost all the cells in neurospheres kept alive (Figure
1D). Asymmetric division experiments demonstrated that newly formed neurospheres grew in suspension and were maintained as spheres in stem cell medium; however, adherent growth and morphology was induced with 10% FBS administration (Figure
1E).GL261 and U87 cells, as well as primary glioma cells isolated from surgical waste were used as glioma cell lines. Magnetic cell sorting (MACS; Miltenyi Biotech, Bergisch-Gladbach, Germany) was used to isolate CD133−CD15−NESTIN− cells . In brief, primary glioma, GL261 or U87 cells were incubated in DMEM/F12+10% FBS at 37°C for 3 days, and cell suspensions were prepared following 0.25% trypsin digestion. The cells were subsequently counted and re-suspended in PBS that contained 0.08% EDTA and 0.5% BSA (PBSE; 108 cells/500 μl), followed by incubation for 15 min at 4°C with polyclonal rabbit anti-human CD133+ IgGs (Miltenyi Biotech, Germany) or polyclonal rabbit anti-mouse CD133+ IgGs (Miltenyi Biotech, Germany). After washing with PBS that contained 1% BSA, the cells were re-suspended in PBSE (108 cells/300 μl), labeled with goat anti-rabbit IgG MicroBeads (Miltenyi Biotech, Germany), incubated for 15 min at 10°C, washed twice with PBSE and re-suspended in 500 μl PBSE. A cell separation column with a flow resistor was subsequently placed in a miniMACS magnet fixed on the MACS multistand and flushed with 500 μl PBSE. The 500 μl cell suspension was poured into the column reservoir. The CD133+ cells were retained within the magnet, and unlabeled nonmagnetic CD133− cells passed through the column and were collected. To increase the purity of the CD133− cells, we repeated the previous steps three times. The same methods were used to sort CD15− cells from CD133− cells and NESTIN− cells from CD133−CD15− cells. The CD133−CD15−NESTIN− cells were subsequently cultured in DMEM/F12+10% FBS at 37°C to maintain their differentiated status.We would like to thank Prof. Jiqiang Zhang (Neurobiology, Third Military Medical University) for his constructive suggestions in the preparation of this manuscript. This work was supported by the National Natural Science Foundation of China (NSFC 81272784) and the Science Foundation of Southwest Hospital (SWH2015QN10).CONFLICTS OF INTERESTThe authors declare there are no potential conflicts of interest.