title: 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.
authors.affiliation: Department of Surgery A, Heinrich-Heine University and University Hospital Düsseldorf, Düsseldorf, Germany.
authors.forename: Anna C
authors.initials: AC
authors.lastname: Luca
authors.forename: Sabrina
authors.initials: S
authors.lastname: Mersch
authors.forename: René
authors.initials: R
authors.lastname: Deenen
authors.forename: Stephan
authors.initials: S
authors.lastname: Schmidt
authors.forename: Isabelle
authors.initials: I
authors.lastname: Messner
authors.forename: Karl-Ludwig
authors.initials: KL
authors.lastname: Schäfer
authors.forename: Stephan E
authors.initials: SE
authors.lastname: Baldus
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Huckenbeck
authors.forename: Roland P
authors.initials: RP
authors.lastname: Piekorz
authors.forename: Wolfram T
authors.initials: WT
authors.lastname: Knoefel
authors.forename: Andreas
authors.initials: A
authors.lastname: Krieg
authors.forename: Nikolas H
authors.initials: NH
authors.lastname: Stoecklein
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000473:pathology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005109:Extracellular Matrix / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007797:Laminin / Q000378:metabolism; D010641:Phenotype; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000187:drug effects / Q000473:pathology; D059016:Tumor Microenvironment / Q000187:drug effects
chemical_list: D007797:Laminin; D047428:Protein Kinase Inhibitors; C512478:EGFR protein, human; D066246:ErbB Receptors
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