In vitro ovarian cancer model based on three-dimensional agarose hydrogel

Abstract

To establish a typical tumor model of ovarian cancer which may be more representative and reliable than traditional monolayer culture and pellet, agarose was used as cell vehicle to engineering tumor. Selection of agarose is based on its successful application in tissue engineering with both amenable mechanical and biological properties. In this study, ovarian cancer cell line SKOV3 was encapsulated in agarose hydrogel with cell aggregates and two-dimensional culture as controls. In vitro cell proliferation was assessed by MTT and cell viability was examined at time points of 2, 4, and 6 days. The expression of tumor malignancy markers including matrix metalloproteinase 2, matrix metalloproteinase 9, hypoxia-inducible factor-1α, and vascular endothelial growth factor-A was assessed by real-time polymerase chain reaction. The results showed that cells proliferated more rapidly in three-dimensional agarose culture than controls. Furthermore, upregulation of matrix metalloproteinase 9 and matrix metalloproteinase 2 activity and increased expression of vascular endothelial growth factor-A and hypoxia-inducible factor-1α were shown in agarose-engineered tumors. All the evidences demonstrated that agarose may provide a more favorable environment for cancer cell growth, mimicking the in vivo environment for tumor generation. The novel in vitro tumor model may be useful for the further investigation of anticancer therapeutics.

Keywords: Agarose; ovarian cancer; tumor engineering; tumor microenvironment.

Figure 1.

MTT was used to analyze cell proliferation in 3D and 2D cultures. The cell proliferation in 3D agarose was higher than pellets and 2D monolayer at 2, 4, and 6 days. 2D culture showed the lowest cell numbers among the groups. */#, **/##, and ***/### denote p < 0.05, p < 0.01, and p < 0.001, respectively. MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 3D: three-dimensional; 2D: two-dimensional.

Figure 2.

Cell viability in the agarose scaffold materials and in control groups visualized using confocal laser scanning microscopy and Live/Dead cell assay kit: (a) 2D control, (b) 3D control, and (c) 3D agarose. Cells in 3D agarose and pellets displayed a spherical morphology, while monolayer culture showed cells of fusiform morphology; proliferation in 3D agarose scaffolds is more rapid than the 3D control group. As limited by space, the most notable cell death was shown in monolayer culture. Scale bar is 100 µm. 2D: two-dimensional; 3D: three-dimensional.

Figure 3.

Morphology of ovarian cancer cells was observed after 2, 4, and 6, days of culture in agarose, pellets, and monolayer: (a) 2D control, (b) 3D control, and (c) 3D agarose. More cell numbers, more notable cell growth, as well as more matrix were presented in 3D agarose culture than in pellets culture. Obvious cell clusters was observed in 3D agarose. Scale bar is 100 µm. Arrows indicate the clumps of cells in 3D agarose after 2, 4, and 6 days of culture. 2D: two-dimensional; 3D: three-dimensional.

Figure 4.

Quantitative RT-PCR was used to analyze the progression of MMP-2 and MMP-9 gene expression in both 3D and 2D cultures over a 6-day period: (a) MMP-2 and (b) MMP-9 were greatly upregulated in 3D agarose compared to the control groups, on days 2, 4, and 6. */#, **/##, and ***/### denote p < 0.05, p < 0.01, and p < 0.001, respectively. RT-PCR: reverse transcription polymerase chain reaction; MMP: matrix metalloproteinase; 3D: three-dimensional; 2D: two-dimensional.

Figure 5.

HIF-1α gene expression was continuously increased over time in both 3D and pellets, in comparison to monolayer culture with little change. The expression of HIF-1α was much higher in 3D agarose than in pellets and 2D culture. */#, **/##, and ***/### denote p < 0.05, p < 0.01, and p < 0.001, respectively. HIF: hypoxia-inducible factor; 3D: three-dimensional; 2D: two-dimensional.

Figure 6.

In correlation with HIF-1α, VEGF-A exhibited sustained increase with time in both 3D and pellets. There is a little change in VEGF-A expression in monolayer culture. The expression of VEGF-A was much higher in 3D agarose than in pellets and 2D culture. */#, **/##, and ***/### denote p < 0.05, p < 0.01, and p < 0.001, respectively. HIF: hypoxia-inducible factor; VEGF: vascular endothelial growth factor–A; 3D: three-dimensional; 2D: two-dimensional.