Project description:It is increasingly appreciated that properties of cultured epithelial cells differ dramatically in 2D compared to 3D, and the latter more faithfully recapitulates in vivo behavior. By studying a battery of human colorectal cancer (CRC) cell lines in type-1 collagen, we have found that HCA-7 cells form colonies with two distinctive and persistent morphological and functional properties. We observed predominantly single-layered polarized cysts (cystic colonies, CC) and a smaller fraction displaying disorganized solid masses (spiky colonies, SC) that were highly invasive in vivo. Despite overall genomic similarity, CC and SC exhibited distinct and dynamic patterns of gene expression in 3D.
Project description:It is increasingly appreciated that properties of cultured epithelial cells differ dramatically in 2D compared to 3D, and the latter more faithfully recapitulates in vivo behavior. By studying a battery of human colorectal cancer (CRC) cell lines in type-1 collagen, we have found that HCA-7 cells form colonies with two distinctive and persistent morphological and functional properties. We observed predominantly single-layered polarized cysts (cystic colonies, CC) and a smaller fraction displaying disorganized solid masses (spiky colonies, SC) that were highly invasive in vivo. Despite overall genomic similarity, CC and SC exhibited distinct and dynamic patterns of gene expression in 3D.
Project description:We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.
Project description:We identified a subpopulation of macrophages that are specifically enriched in interstitial lung disease. To better understand the functional relevance of this population with regards to disease pathogenesis by manipulating them in vitro, we attempted to induce this state in cell culture by prepurposing a published 3D hydrogel culture system originally designed to generate hematopoietic progenitor cells from PBMCs.
Project description:We examined whether SATB1 functions as a global gene regulator in order to maintain the aggressive phenotype of the MDA-MB-231 cell line. We compared the gene expression profiles between control_shRNA-MDA-MB-231 cells, which express SATB1 at high levels, and SATB1_shRNA1-MDA-MB-231 in which the level of SATB1 was greatly downregulated by RNAi technology. This comparative studies were performed using two different platforms (Codelink and Affymetrix genechip) with two culture conditions either on plastic dish (2D) or on matrigel (3D) which allows cells to form a breast-like morphology only for non-aggressive cells. Keywords: Comparative studies on Control_shRNA and SATB1_shRNA1 expressing MDA-MB-231 from 2D or 3D culture. We examined control_shRNA-MDA-MB-231 cells and SATB1_shRNA1-MDA-MB-231 cells under two culture condition;on plastic dish(2D culture) and on Matrigel coated dish(3D culture). When SATB1 was depleted by RNAi technology, these normally aggressive cells exhibited normal breast like morphology on 3D. We used two different microarray platforms (Codelink and Affymetrix) to make expression data. Initial analysis of data and cross-platform comparison were performed using Codelink expression analysis and GeneSpring software. We provide ratio for control_shRNA/SATB1_shRNA1-MDA-MB-231 cells for 2D and 3D on this series.
Project description:Preclinical cancer drug discovery efforts have employed two-dimensional (2D)-cell-based assay models, which fail to forecast in vivo efficacy and contribute to a lower success rates of clinical approval. Three-dimensional (3D) cell culture models are recently expected to bridge the gap between 2D and in vivo models. We have developed a novel 3D culture method that is suitable for imaging analysis and improves the growth of spheroid-forming cancer cells under anchorage-independent condition by leveraging a feature of LA717, a seaweed-derived polysaccharide. Gene microarrays were used to observe the global gene expression in A549 cells cultured with adhesion condition (2D, control) or with low adhesion condition (LA717) and identified distinct classes of up or down-regulated genes.