Department of Engineering and System Science, National Tsing Hua University, Hsinchu, Taiwan.
Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan.
PLoS One. 2020 Nov 11;15(11):e0240833. doi: 10.1371/journal.pone.0240833. eCollection 2020.
Vascular endothelial growth factor (VEGF) is a major cytokine in tumor biology affecting tumor survival, aggressiveness and pro-angiogenetic activities. In addition, cellular stresses often result in aggressive pro-angiogenetic behavior in tumors. For in vitro study, conventional monolayer cell culture has been broadly exploited; however, it often provides limited information due to its different microenvironment from that in vivo. Recently, three-dimensional (3D) cell spheroid culture provides in vivo-like microenvironments to study tumor biology and their survival mechanisms with better predictive power. In this work, vascular endothelial growth factor of type A (VEGF-A) secretion from osteosarcoma (MG-63) cells cultured using monolayer and 3D spheroid models under two stress conditions: nutrient deficiency (reduced serum culture) and hypoxia-inducible factor (HIF) inhibition (HIF inhibitor, YC-1) are characterized and systematically compared. In order to obtain ample sample size for consistent characterization of cellular responses from cancer spheroids under the stresses and compare the responses to those from the conventional monolayer model, a microfluidic spheroid formation and culture device is utilized in the experiments. In the analysis, cell viability is estimated from captured images, and quantification of VEGF-A secreted from the cells is achieved using enzyme-linked immunosorbent assay (ELISA). The experimental results show that the viabilities decrease when the cells face higher stress levels in both monolayer and 3D spheroid culture models; however, the VEGF-A secretion profiles between the cell culture models are different. The VEGF-A secretion decreases when the cells face higher stress conditions in the monolayer cell culture. In contrast, for the 3D spheroid culture, the VEGF-A concentration decreases for low stress levels but increases while the stress level is high. The VEGF-A regulation in the 3D models mimics in vivo cases of tumor survival and can provide insightful information to investigate tumor angiogenesis in vitro. The approach developed in this paper provides an efficient method to quantitatively and statistically study tumor growth kinetics and stress responses from highly uniform samples and it can also be applied to compare the underlying biomolecular mechanisms in monolayer and 3D spheroid culture models to elucidate the effects of microenvironments on cellular response in cancer research.
血管内皮生长因子（VEGF）是肿瘤生物学中的一种主要细胞因子，影响肿瘤的存活、侵袭性和促血管生成活性。此外，细胞应激通常会导致肿瘤中侵袭性的促血管生成行为。在体外研究中，传统的单层细胞培养得到了广泛的应用；然而，由于其与体内不同的微环境，它通常提供的信息有限。最近，三维（3D）细胞球体培养为研究肿瘤生物学及其生存机制提供了类似于体内的微环境，具有更好的预测能力。在这项工作中，我们研究了在两种应激条件下（营养缺乏（减少血清培养）和缺氧诱导因子（HIF）抑制（HIF 抑制剂，YC-1）），使用单层和 3D 球体模型培养的骨肉瘤（MG-63）细胞中血管内皮生长因子 A（VEGF-A）的分泌，并对其进行了系统比较。为了获得足够的样本量，以一致地表征应激下癌细胞球体的细胞反应，并比较其与传统单层模型的反应，我们在实验中使用了微流控球体形成和培养装置。在分析中，从捕获的图像中估计细胞活力，并用酶联免疫吸附测定（ELISA）定量测定细胞分泌的 VEGF-A。实验结果表明，在单层和 3D 球体培养模型中，当细胞面临更高的应激水平时，细胞活力都会下降；然而，细胞培养模型之间的 VEGF-A 分泌模式不同。在单层细胞培养中，当细胞面临更高的应激条件时，VEGF-A 的分泌减少。相比之下，对于 3D 球体培养，当应激水平较低时，VEGF-A 的浓度会降低，但当应激水平较高时，VEGF-A 的浓度会增加。3D 模型中的 VEGF-A 调节模拟了体内肿瘤存活的情况，可以提供有价值的信息来研究体外肿瘤血管生成。本文所开发的方法提供了一种有效的方法，可以从高度均匀的样本中定量和统计地研究肿瘤生长动力学和应激反应，还可以应用于比较单层和 3D 球体培养模型中的潜在生物分子机制，以阐明微环境对癌症研究中细胞反应的影响。
