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The Impact of Astrocytes and Endothelial Cells on Glioblastoma Stemness Marker Expression in Multicellular Spheroids

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Abstract

Introduction

Glioblastoma multiforme (GBM), the most common primary brain tumor in adults, is extremely malignant and lethal. GBM tumors are highly heterogenous, being comprised of cellular and matrix components, which contribute to tumor cell invasion, cancer stem cell maintenance, and drug resistance. Here, we developed a heterotypic 3D spheroid model integrating GBM cells with astrocytes and endothelial cells (ECs) to better simulate the cellular components of the tumor microenvironment and investigate their impact on the stemness marker expression of GBM cells, which has not been previously investigated.

Methods

We used U87 GBM cells, C8-D1A mouse astrocytes, and human umbilical vein ECs to construct co- and tri-culture spheroid models in low-attachment U-well plates. We characterized the expression of known stemness markers NESTIN, SOX2, CD133, NANOG, and OCT4 in these models and compared it to respective mixed monoculture spheroids (control) using qRT-PCR and immunostaining.

Results

We incorporated GBM cells and astrocytes/ECs in 1:1, 1:2, 1:4, and 1:9 ratio and observed spontaneous self-assembled spheroids in all coculture conditions. We observed changing spheroid size dynamics over 7 days and an increased expression in stemness markers in GBM-astrocyte and GBM-EC coculture spheroids in 1:4 and 1:9 coculture conditions, respectively. In a triculture model employing GBM cells, astrocytes, and ECs in a 1:4:9 ratio, we found an increased expression of all the stemness markers.

Conclusions

We elucidated the impact of astrocytes and ECs on GBM stemness marker expression. This multicellular spheroid model may provide an important tool for investigating the crosstalk between cell types in GBM.

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Acknowledgments

This work was supported by the National Science Foundation (CBET 1604677) and the Alabama EPSCoR Graduate Research Fellowship (P.N.). The authors thank Dr. Yu** Bao (University of Alabama) for providing the C8D1A astrocytes and Dr. Matthew Jenny (University of Alabama) for the use of his Nanodrop 2000C spectrophotometer.

Conflict of interest

P. S. Nakod, Y. Kim, and S. S. Rao declare that they have no conflicts of interest.

Ethical Approval

No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.

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  1. Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL, 35487, USA

    Pinaki S. Nakod, Yonghyun Kim & Shreyas S. Rao

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Correspondence to Shreyas S. Rao.

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Nakod, P.S., Kim, Y. & Rao, S.S. The Impact of Astrocytes and Endothelial Cells on Glioblastoma Stemness Marker Expression in Multicellular Spheroids. Cel. Mol. Bioeng. 14, 639–651 (2021). https://doi.org/10.1007/s12195-021-00691-y

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