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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.

SUBMITTER: Sarkar S

PROVIDER: S-EPMC7657494 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Sarkar Sreerupa S Peng Chien-Chung CC Tung Yi-Chung YC

PloS one 20201111 11

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 ...[more]

PMID: 33175874

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Project description:Background:Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. Methods:We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. Results:The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. Conclusion:These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

Project description:Most patients who succumb to cancer have metastases to bone that contribute to their death. Cancer cells that metastasize to bone are regularly subjected to mechanical stimuli that may affect their proliferation, growth and protein expression. Understanding why some cancer cells thrive in this environment could provide insight into new approaches to prevent or treat metastasis to bone. We used 4T1 cells as a model of breast cancer cells, and implanted them in gelatin hydrogels with moduli of 1 or 2.7 kPa to mimic the properties of bone marrow. The constructs were subjected to either perfusion of media through the hydrogel or combined perfusion and cyclic mechanical compression for 1 h d-1 for 4 d. Controls were cultured in free-swelling conditions. The cells formed spheroids during the 4 d of culture, with larger spheroids in the statically cultured constructs than in perfusion or compressed constructs. In stiffer gelatin, smaller spheroids formed in compressed constructs than perfusion alone, while compression had no effect compared to perfusion in the softer gelatin. Immunostaining indicated that the spheroids expressed osteopontin, parathyroid hormone-related protein and fibronectin, which are all hallmarks of bone metastasis. The proliferative marker Ki67 was present in all spheroids on day 4. In the 1 kPa gelatin, Ki67 staining intensity was greater in the statically cultured, free-swelling constructs than in bioreactor culture, regardless of dynamic compression. By contrast, proliferation was higher in the compressed gelatins compared to perfusion alone in the 2.7 kPa constructs, although the spheroids were smaller, on average. This suggests the stiffer gelatin may restrict spheroid growth at the same time that it enhances mechanobiological signalling during compression. Taken together, 4T1 breast cancer cells are mechanically sensitive, and mechanical stimuli can alter their proliferation and protein expression within soft materials with mechanical properties similar to bone marrow. As such, both in vivo and in vitro models of cancer metastasis should consider the role of the mechanical environment in the bone.

Dataset Information

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

Publications

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

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