Skip to main content

Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma


Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5 % fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5 % FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5 % FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


  1. 1.

    Oike T, Suzuki Y, K-i S, Shirai K, S-e N, Tamaki T, Nagaishi M, Yokoo H, Nakazato Y, Nakano T (2013) Radiotherapy plus concomitant adjuvant temozolomide for glioblastoma: Japanese mono-institutional results. PLoS One 8(11):e78943

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Sengupta S, Marrinan J, Frishman C, Sampath P (2012) Impact of temozolomide on immune response during malignant glioma chemotherapy. Clin Dev Immunol 2012

  3. 3.

    Shen G, Shen F, Shi Z, Liu W, Hu W, Zheng X, Wen L, Yang X (2008) Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods. Vitro Cellular & Developmental Biology-Animal 44(7):280–289

    CAS  Article  Google Scholar 

  4. 4.

    Knizetova P, Ehrmann J, Hlobilkova A, Vancova I, Kalita O, Kolar Z, Bartek J (2008) Autocrine regulation of glioblastoma cell-cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay. Cell Cycle 7(16):2553–2561

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Bachelder RE, Wendt MA, Mercurio AM (2002) Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4. Cancer Res 62(24):7203–7206

    CAS  PubMed  Google Scholar 

  6. 6.

    Hamerlik P, Lathia JD, Rasmussen R, Wu Q, Bartkova J, Lee M, Moudry P, Bartek J, Fischer W, Lukas J (2012) Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth. J Exp Med 209(3):507–520

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Li P, Zhou C, Xu L, Xiao H (2013) Hypoxia enhances stemness of cancer stem cells in glioblastoma: an in vitro study. Int J Med Sci 10(4):399–407

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Karamboulas C, Ailles L (2013) Developmental signaling pathways in cancer stem cells of solid tumors. Biochimica et Biophysica Acta (BBA)-General Subjects 1830(2):2481–2495

    CAS  Article  Google Scholar 

  9. 9.

    Swamydas M, Ricci K, Rego SL, Dréau D (2013) Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases. Cell Adhes Migr 7(3):315–324

    Article  Google Scholar 

  10. 10.

    Zhou X, Wang X, Qu F, Zhong Y, Lu X, Zhao P, Wang D, Huang Q, Zhang L, Li X (2009) Detection of cancer stem cells from the C6 glioma cell line. J Int Med Res 37(2):503–510

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Ye X-Q, Wang G-H, Huang G-J, Bian X-W, Qian G-S, Yu S-C (2011) Heterogeneity of mitochondrial membrane potential: a novel tool to isolate and identify cancer stem cells from a tumor mass? Stem Cell Rev Rep 7(1):153–160

    CAS  Article  Google Scholar 

  12. 12.

    Yao K, Gietema J, Shida S, Selvakumaran M, Fonrose X, Haas N, Testa J, O’Dwyer P (2005) In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo. Br J Cancer 93(12):1356–1363

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Strasser U, Fischer G (1995) Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation. J Neurosci Methods 57(2):177–186

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Cimarosti H, Rodnight R, Tavares A, Paiva R, Valentim L, Rocha E, Salbego C (2001) An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation. Neurosci Lett 315(1):33–36

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Keith B, Simon MC (2007) Hypoxia-inducible factors, stem cells, and cancer. Cell 129(3):465–472

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S, Shi Q, Cao Y, Lathia J, McLendon RE (2009) Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell 15(6):501–513

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Zagzag D, Lukyanov Y, Lan L, Ali MA, Esencay M, Mendez O, Yee H, Voura EB, Newcomb EW (2006) Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion. Lab Investig 86(12):1221–1232

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Axelson H, Fredlund E, Ovenberger M, Landberg G, Påhlman S (2005) Hypoxia-induced dedifferentiation of tumor cells—a mechanism behind heterogeneity and aggressiveness of solid tumors. In: Seminars in cell & developmental biology, vol 4. Elsevier, pp. 554–563

  19. 19.

    Nagaraj NS, Vigneswaran N, Zacharias W (2004) Hypoxia-mediated apoptosis in oral carcinoma cells occurs via two independent pathways. Mol Cancer 3(1):1

    Article  Google Scholar 

  20. 20.

    Harris AL (2002) Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer 2(1):38–47

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Weinmann M, Jendrossek V, Handrick R, Güner D, Goecke B, Belka C (2004) Molecular ordering of hypoxia-induced apoptosis: critical involvement of the mitochondrial death pathway in a FADD/caspase-8 independent manner. Oncogene 23(21):3757–3769

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Leszczynska KB, Foskolou IP, Abraham AG, Anbalagan S, Tellier C, Haider S, Span PN, O’Neill EE, Buffa FM, Hammond EM (2015) Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT. J Clin Invest 125(6):2385–2398

    Article  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Asai A, Miyagi Y, Sugiyama A, Gamanuma M, Hong SI, Takamoto S, Nomura K, Matsutani M, Takakura K, Kuchino Y (1994) Negative effects of wild-type p53 and s-Myc on cellular growth and tumorigenicity of glioma cells. J Neuro-Oncol 19(3):259–268

    CAS  Article  Google Scholar 

  24. 24.

    Zenali MJ, Tan D, Li W, Dhingra S, Brown RE (2010) Stemness characteristics of fibrolamellar hepatocellular carcinoma: immunohistochemical analysis with comparisons to conventional hepatocellular carcinoma. Annals of Clinical & Laboratory Science 40(2):126–134

    Google Scholar 

  25. 25.

    Li Q, Rycaj K, Chen X, Tang DG (2015) Cancer stem cells and cell size: a causal link? In: Seminars in cancer biology. Elsevier, pp. 191–199

  26. 26.

    Murayama A, Matsuzaki Y, Kawaguchi A, Shimazaki T, Okano H (2002) Flow cytometric analysis of neural stem cells in the developing and adult mouse brain. J Neurosci Res 69(6):837–847

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF (2001) Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 412(6848):736–739

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Narayanan G, Poonepalli A, Chen J, Sankaran S, Hariharan S, Yu YH, Robson P, Yang H, Ahmed S (2012) Single-cell mRNA profiling identifies progenitor subclasses in neurospheres. Stem Cells Dev 21(18):3351–3362

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    J-j D, Qiu W, Xu S-l, Wang B, X-z Y, Y-f P, Zhang X, X-w B, Yu S-c (2013) Strategies for isolating and enriching cancer stem cells: well begun is half done. Stem Cells Dev 22(16):2221–2239

    Article  Google Scholar 

  30. 30.

    Zheng X, Shen G, Yang X, Liu W (2007) Most C6 cells are cancer stem cells: evidence from clonal and population analyses. Cancer Res 67(8):3691–3697

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Michelakis E, Sutendra G, Dromparis P, Webster L, Haromy A, Niven E, Maguire C, Gammer T-L, Mackey J, Fulton D (2010) Metabolic modulation of glioblastoma with dichloroacetate. Sci Transl Med 2(31):31ra34

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Schieke SM, Ma M, Cao L, McCoy JP, Liu C, Hensel NF, Barrett AJ, Boehm M, Finkel T (2008) Mitochondrial metabolism modulates differentiation and teratoma formation capacity in mouse embryonic stem cells. J Biol Chem 283(42):28506–28512

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Seton-Rogers S (2011) Cancer stem cells: VEGF promotes stemness. Nat Rev Cancer 11(12):831–831

    CAS  PubMed  Google Scholar 

  34. 34.

    Yuan X, Curtin J, Xiong Y, Liu G, Waschsmann-Hogiu S, Farkas DL, Black KL, John SY (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23(58):9392–9400

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64(19):7011–7021

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Sakaki T, Yamada K, Otsuki H, Yuguchi T, Kohmura E, Hayakawa T (1995) Brief exposure to hypoxia induces bFGF mRNA and protein and protects rat cortical neurons from prolonged hypoxic stress. Neurosci Res 23(3):289–296

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Kyurkchiev D (2014) Cancer stem cells from glioblastoma multiforme: culturing and phenotype. Stem Cells 2(1):3

    Google Scholar 

  38. 38.

    Freyhaus H, Dagnell M, Leuchs M, Vantler M, Berghausen EM, Caglayan E, Weissmann N, Dahal BK, Schermuly RT, Östman A (2011) Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases. Am J Respir Crit Care Med 183(8):1092–1102

    Article  PubMed  Google Scholar 

  39. 39.

    Segovia J, Lawless GM, Tillakaratne NJ, Brenner M, Tobin AJ (1994) Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells. J Neurochem 63(4):1218–1225

    CAS  Article  PubMed  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Mariana Maier Gaelzer.

Ethics declarations


This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).

Additional information

Mariana Maier Gaelzer and Mariana Silva dos Santos contributed equally to this work.

Electronic supplementary material

Figure S1

Photomicrographs, propidium iodide (PI) and DAPI staining of C6 cells exposed to OD in 5 % FBS media (n = 6). (GIF 137 kb)

Figure S2

Photomicrographs, propidium iodide (PI) and DAPI staining of C6 cells exposed to OD in serum-free media (n = 6). (GIF 98 kb)

Figure S3

Dot-plot analysis of cell size from flow cytometry of C6 cells in (A) 5 % FBS medium or (B) serum-free medium. Cells were stained with annexin V and PI and categorized into three sizes: small [S], medium [M] and large [L]. (GIF 62 kb)

Figure S4

Dot-plot analysis of cell granularity from flow cytometry of C6 cells in (A) 5 % FBS medium or (B) serum-free medium. Cells were stained with Annexin V and PI and categorized based on their granularity: regular [G1], more granular [G2] (GIF 61 kb)

Figure S5

Photomicrographs of C6 cells exposed to various times of OD on serum-free media followed by RO. (GIF 192 kb)

Figure S6

Photomicrographs and sulforhodamine B staining comparing the morphology of C6 cells exposed to 1 h OD in serum-free media and 24 h RO in 5 % fetal bovine serum. (GIF 27 kb)

High resolution (TIFF 1754 kb)

High resolution (TIFF 1535 kb)

High resolution (TIFF 778 kb)

High resolution (TIFF 710 kb)

High resolution (TIFF 996 kb)

High resolution (TIFF 444 kb)

Table S1

Morphological changes of C6 glioma cells after oxygen-deprivation (OD) on serum-free medium and reoxygenation (RO) in 5 % fetal bovine serum. (PDF 19 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gaelzer, M.M., Santos, M.S.d., Coelho, B.P. et al. Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma. Mol Neurobiol 54, 6261–6272 (2017).

Download citation


  • Cancer
  • Glioma
  • Hypoxia
  • Reoxygenation
  • C6
  • Cancer stem cell