Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines

Abstract

Three-dimensional (3D) tumor cell cultures grown in laminin-rich-extracellular matrix (lrECM) are considered to reflect human tumors more realistic as compared to cells grown as monolayer on plastic. Here, we systematically investigated the impact of ECM on phenotype, gene expression, EGFR signaling pathway, and on EGFR inhibition in commonly used colorectal cancer (CRC) cell lines. LrECM on-top (3D) culture assays were performed with the CRC cell lines SW-480, HT-29, DLD-1, LOVO, CACO-2, COLO-205 and COLO-206F. Morphology of lrECM cultivated CRC cell lines was determined by phase contrast and confocal laser scanning fluorescence microscopy. Proliferation of cells was examined by MTT assay, invasive capacity of the cell lines was assayed using Matrigel-coated Boyden chambers, and migratory activity was determined employing the Fence assay. Differential gene expression was analyzed at the transcriptional level by the Agilent array platform. EGFR was inhibited by using the specific small molecule inhibitor AG1478. A specific spheroid growth pattern was observed for all investigated CRC cell lines. DLD-1, HT-29 and SW-480 and CACO-2 exhibited a clear solid tumor cell formation, while LOVO, COLO-205 and COLO-206F were characterized by forming grape-like structures. Although the occurrence of a spheroid morphology did not correlate with an altered migratory, invasive, or proliferative capacity of CRC cell lines, gene expression was clearly altered in cells grown on lrECM as compared to 2D cultures. Interestingly, in KRAS wild-type cell lines, inhibition of EGFR was less effective in lrECM (3D) cultures as compared to 2D cell cultures. Thus, comparing both 2D and 3D cell culture models, our data support the influence of the ECM on cancer growth. Compared to conventional 2D cell culture, the lrECM (3D) cell culture model offers the opportunity to investigate permanent CRC cell lines under more physiological conditions, i.e. in the context of molecular therapeutic targets and their pharmacological inhibition.

Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors

“…33 For example, Luca et al noted that among the CRC cell lines they tested, those derived from metastatic cells formed grape-like morphology in lrECM matrix, whereas CRC cell lines established from primary tumor tissue formed mass spheroid morphology. 29 They also found that spheroid morphology did not correlate with the migratory, invasive, or proliferative capacity of CRC cell lines. Hongisto et al 33 reported that the JIMT1 breast cancer cell line formed mass-shaped spheroids on both Matrigel and polyHEMA (poly(2-hydroxyethyl methacrylate)) matrix, but more uniformly shaped and sized spheroids on Matrigel were observed, while more single cells mixed with larger structures appeared on polyHEMA.…”

“…A variety of cell lines showed reduced proliferation rate in 3D cultures compared to those cultured in 2D. 17,[27][28][29][30][31] 33 But interestingly, the same cell line cultured on the synthetic poly(2-hydroxyethylmethacrylate) (polyHEMA) 3D scaffold, exhibited a proliferation rate that was 7.2-fold slower than in 2D culture, 33 which suggests that the cell proliferation rate is also related to the type of 3D model in which the cells are cultured. In general, the proliferation rate of cells grown in 3D culture better represent the growth of tumors in vivo compared to those cultured in an unnatural 2D environment.…”

“…17 Most studies have shown that the cell viability in 3D cultures during the first few days (1-5 days) was not significantly different when compared to that in 2D culture. 30,34 In some cases, 3D cultures showed slightly reduced cell viability 29 when the culture time prolonged, and this was related to the structure of spheroids, which may result in the lack of oxygen and nutrients, and the accumulation of waste at the core of the spheroid as they grow larger. 25 The structures of 3D spheroids.…”

“…38 Overall, the morphological appearance of 3D spheroids is primarily cell line dependent. 29,35,37 However, the type of 3D model utilized may also affect cellular morphology. 33 For example, Luca et al noted that among the CRC cell lines they tested, those derived from metastatic cells formed grape-like morphology in lrECM matrix, whereas CRC cell lines established from primary tumor tissue formed mass spheroid morphology.…”

“…Kiss et al (2013) recently demonstrated that cell-ECM interactions in 3D culture (on Matrigel) can modulate cell morphology and upregulate CXCR4 and CXCR7 expression in prostate cancer cell lines PC3, DU145, and LNCaP. 42 Luca et al 29 reported the first systematic analysis of how the ECM in 3D cultures influence the phenotype and genotype of commonly used colorectal cancer (CRC) cell lines, including CACO-2, DLD-1, HT-29, SW480, LOVO, and COLO-206F. They investigated the expression levels of the epidermal growth factor receptor (EGFR) and the downstream activated kinases AKT (also known as protein kinase B) and p42/44 MAPK (mitogenactivated protein kinases), since EGFR stimulates proliferation via MAP-kinases, which has been established as a therapeutic target in the treatment of advanced CRC.…”