Comparison of VEGF-A secretion from tumor cells under cellular stresses in conventional monolayer culture and microfluidic three-dimensional spheroid models

Abstract

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.

In vitro three-dimensional cell cultures for bone sarcomas

“…Massive production of spheroids was used to challenge spheroids to two different cellular stresses: nutrient deprivation (serum concentration) and hypoxia (HIF inhibition). 3D cultures obtained data confirmed in vivo observation where stress conditions favoured the increase of VEGF secretion and induction of malignancy processes [99] . This study confirmed the impact of ECM variation in tumour malignancy as well as demonstrated that microfluidic approaches represent an interesting tool for massive 3D cultures and analysis.…”

“…3 ). A microfluidic chip was developed for the production of OS spheroids in mass (up to 5000) [99] . Microfluidic device was treated with a surfactant (Synperonic®) to generate a non-adherent surface that favoured the generation of MG63 spheroids in a similar way as non-adherent plates.…”

Exosomal LBH inhibits epithelial-mesenchymal transition and angiogenesis in nasopharyngeal carcinoma via downregulating VEGFA signaling

“…Then the same tissues were subjected to western blotting, and we found that LBH upregulation significantly decreased the protein expression of Vimentin, p-CRYAB and CRYAB, and increased E-cadherin expression (Figure 1C), showing inhibited EMT progression, which is consistent with our previous results in NPC cells. LBH upregulation also significantly decreased VEGFA expression, which was verified by whole-slide images of anti-VEGFA staining (Figure 1A, C), whose upregulation and secretion were generally considered angiogenic [32,33]. Moreover, H&E staining indicated that for both CNE2 and SUNE1 NPC xenografts, the tissues of LBH-overexpressing tumors were well-differentiated compared to the controls, exhibiting relatively epithelial characteristics, while the tissues of Lv5NC tumors showed poorly-differentiated, relatively mesenchymal characteristics (Figure S5).…”

Osteosarcoma tumor microenvironment: the key for the successful development of biologically relevant 3D in vitro models

“…These studies mostly focused on the influence of molecular gradients in cell morphology, or the adhesive properties of OS cells under shear flow or at various temperatures/pHs. In one study, the secretion of VEGF type A from MG63 cells was investigated [100]. OS cells were cultured either in a monolayer or in a spheroid model fabricated in microfluidic chips.…”

“…The results demonstrated lower cell viability under higher stress levels (due to inhibition of HIF and decreased nutrient supply) in both cell culture models. However, in monoculture, the secretion of VEGF decreased in stressful conditions, which does not correspond to the in vivo situation [100]. In another study, a microfluidic platform was developed to test the efficacy of methotrexate and methotrexate-loaded nanoparticles [146].…”