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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.

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Correspondence to Mariana Maier Gaelzer.

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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).

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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)

High resolution (TIFF 1754 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)

High resolution (TIFF 1535 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)

High resolution (TIFF 778 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)

High resolution (TIFF 710 kb)

Figure S5

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

High resolution (TIFF 996 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 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)

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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).

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