Journal
CELLULAR AND MOLECULAR BIOENGINEERING
Volume 14, Issue 6, Pages 639-651Publisher
SPRINGER
DOI: 10.1007/s12195-021-00691-y
Keywords
Brain cancer; Cancer stem cells; Coculture; Triculture; Spheroids
Funding
- National Science Foundation [CBET 1604677]
- Alabama EPSCoR Graduate Research Fellowship
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By constructing a heterotypic 3D spheroid model, we examined the influence of astrocytes and ECs on the expression of stemness markers in GBM. This model may serve as a valuable tool for studying the crosstalk between different cell types in GBM.
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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