Colon cancer cells adopt an invasive phenotype without mesenchymal transition in 3-D but not 2-D culture upon combined stimulation with EGF and crypt growth factors. BMC Cancer 2013 May 02;13:221
Date
05/04/2013Pubmed ID
23638973Pubmed Central ID
PMC3667045DOI
10.1186/1471-2407-13-221Scopus ID
2-s2.0-84876821077 (requires institutional sign-in at Scopus site) 22 CitationsAbstract
BACKGROUND: The intestinal crypt homeostasis is maintained by a combination of growth factors including Wnt, R-Spondin1, Noggin and the epidermal growth factor (EGF). In human colorectal cancer, the Wnt pathway is constitutively activated through genetic and epigenetic alterations in as many as 11 genes encoding components of this crypt stem-cell maintenance mechanism. Although the proliferation of colon cancer cells does not require Wnt, it is possible that colon cancer cells can still respond to the crypt growth factors in the colonic microenvironment. A number of studies have shown that epithelial cells behave differently in 3-D versus 2-D cultures. Because the 3-D conditions more closely mimic the in vivo environment, we examined the effects of Wnt and other crypt growth factors on colon cancer cell growth in 3-D culture.
METHODS: Colon cancer cells were grown in 3-D matrigel supplemented with different combinations of crypt growth factors and colonies were examined for morphology and pathways.
RESULTS: When colon cancer cells were cultured in 3-D with EGF, they grew as round spheroid colonies. However, colon cancer cells also grew as flat, disc-like colonies when cultured with EGF plus Wnt, R-Spondin1 and Noggin. Disc colonies were found to have comparable levels of E-cadherin as the spheroid colonies, but showed decreased E-cadherin at the cell-matrix contact sites. Disc colonies also elaborated F-actin rich protrusions (FRP) at the cell-matrix edge, reminiscent of an invasive phenotype but without the expression of vimentin. These E-cadherin and F-actin alterations were not induced by the four growth factors in 2-D culture. Formation of the disc colonies was inhibited by the knockdown of β-catenin and by protein kinase inhibitors such as gefitinib, imatinib and MK-2206. Furthermore, withdrawal of the crypt growth factors was able to revert the disc colonies to spheroid growth, showing that the invasive phenotype was reversible dependent on the availability of growth factors.
CONCLUSIONS: These findings show that colon cancer cells remain responsive to the growth factors in the crypt microenvironment and can be induced to undergo morphological transformation in the more physiologically relevant 3-D culture.
Author List
Ludwig K, Tse ES, Wang JYAuthor
Kirk A. Ludwig MD Chief, Professor in the Surgery department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Blotting, WesternCell Culture Techniques
Cell Proliferation
Colonic Neoplasms
Epithelial-Mesenchymal Transition
Fluorescent Antibody Technique
HCT116 Cells
HT29 Cells
Humans
Intercellular Signaling Peptides and Proteins
Microscopy, Confocal
Phenotype
Signal Transduction
