Abstract
Simple SummaryMulti-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based colon cancer cell cultures mimic the hypoxic tumor microenvironment. The simple procedure generates a 3D hypoxic tumor microenvironment comprising defined numbers and densities of colon cancer cells with easily controllable lateral dimensions and a thickness defined by pNFS. This pNFS-based multi-layered colon cancer cell culture system is useful for bioassays, for drug screening, and as a replacement for small animals in testing the effects of a hypoxic tumor microenvironment.Three-dimensional (3D) cancer cell culture systems have been developed to aid the study of molecular mechanisms in cancer development, identify therapeutic targets, and test drug candidates. In this study, we developed a strategy for mimicking the hypoxic tumor microenvironment in a 3D cancer cell culture system using multi-layer, nanofibrous poly(ε-caprolactone) (PCL) scaffold (pNFS)-based cancer cell cultures. We found that human colon cancer cells infiltrated pNFS within 3 days and could be cultured three-dimensionally within the NFS. When incubated in four stacks of 30 µm-thick pNFS for 3 days, colon cancer cells in layer three showed partially reduced entry into the S phase, whereas those in layer four, located farthest from the media, showed a marked reduction in S-phase entry. As a consequence, cells in layer four exhibited hypoxia-induced disorganization of F-actin on day 3, and those in layers three and four showed an increase in the expression of the hypoxia-induced transcription factor HIF-1α and its target genes, Glut1, CA9, VEGF, and LDHA. Consistent with these results, doxorubicin- and ionizing radiation-induced cell death was reduced in colon cancer cells cultured in layers three and four. These results suggest that pNFS-based multi-layer colon cancer cell cultures mimic the hypoxic tumor microenvironment and are useful for bioassays.
Highlights
Three-dimensional (3D) cell culture systems represent the real microenvironment in which cells exist in tissues more accurately than two-dimensional (2D) cell culture systems [1,2,3,4,5,6]
We present a strategy for mimicking the 3D hypoxic tumor microenvironment using a multi-layer pNFS-based colon cancer cell culture system
Proliferating colon cancer cells were detected in the top layer adjacent to the medium, but the number of dividing cells decreased as the distance from the medium increased (Figure 2D and Supplementary Figure S2). These results suggest that cancer cells in layer 3 (L3) and layer 4 (L4) became hypoxic on days 2 and 3
Summary
Three-dimensional (3D) cell culture systems represent the real microenvironment in which cells exist in tissues more accurately than two-dimensional (2D) cell culture systems [1,2,3,4,5,6]. It remains difficult to accurately reconstruct the precise 3D locations of many types of cells in such spheroid cultures owing to cellular heterogeneity, the inability to control cell numbers, and necrosis caused by a lack of nutrients [4]. Alternative multi-layered 3D cell culture systems, such as polymer-based mesh, mesh-like hydrogel sheets, paper-supported gels, polystyrene, and hydrogel, have been developed to improve 3D culture systems and mimic tumor microenvironments [1,13,14,15,16,17,18]. Cells in each layer are separated from each other, making it difficult to detect intercellular interactions between layers; cell density is not as high as the actual density in living tissue; and specific instrumentation that is not commonly available in biological laboratories is often required
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