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.

中文翻译：

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星形胶质细胞和内皮细胞对多细胞球体中胶质母细胞瘤干细胞标记表达的影响

介绍

多形性胶质母细胞瘤 (GBM) 是成人中最常见的原发性脑肿瘤，具有极高的恶性和致命性。GBM 肿瘤具有高度异质性，由细胞和基质成分组成，有助于肿瘤细胞侵袭、癌症干细胞维持和耐药性。在这里，我们开发了一个异型 3D 球体模型，将 GBM 细胞与星形胶质细胞和内皮细胞 (EC) 整合在一起，以更好地模拟肿瘤微环境的细胞成分，并研究它们对 GBM 细胞干性标记表达的影响，这在以前没有被研究过。

方法

我们使用 U87 GBM 细胞、C8-D1A 小鼠星形胶质细胞和人脐静脉 EC 在低附着 U 孔板中构建共培养和三培养球体模型。我们对这些模型中已知干性标记 NESTIN、SOX2、CD133、NANOG 和 OCT4 的表达进行了表征，并使用 qRT-PCR 和免疫染色将其与各自的混合单一培养球体（对照）进行了比较。

结果

我们以 1:1、1:2、1:4 和 1:9 的比例结合了 GBM 细胞和星形胶质细胞/EC，并在所有共培养条件下观察到自发自组装球体。我们观察到 7 天内球体大小动态的变化以及 GBM-星形胶质细胞和 GBM-EC 共培养球体中干性标记的表达增加，分别在 1:4 和 1:9 共培养条件下。在采用 1:4:9 比例的 GBM 细胞、星形胶质细胞和 ECs 的三培养模型中，我们发现所有干性标记的表达增加。

结论

我们阐明了星形胶质细胞和 ECs 对 GBM 干性标记表达的影响。这种多细胞球体模型可能为研究 GBM 中细胞类型之间的串扰提供了重要工具。