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Characterization of HT-29 cells cultured in NanoCulture plates

ABSTRACT: To characterize cells cultured in 3D culture system using NanoCulture plates (NCPs) (Scivax) and in 2D culture, we examined gene expression in HT-29 cells on day 1 or day 7 on NCPs or 2D monolayer culture, using DNA microarrays. From our study, tumour cells grown on NCPs were morphologically different from cells in 2D monolayers, and the cells on NCPs actively migrated and aggregated to form multicellular spheroids (MCSs). To characterize this cell migration and intercellular adhesion, we compared gene expression in HT-29 cells on day 1 on NCPs and 2D monolayer culture using DNA microarrays. We could not find any significant difference in gene expression related to cell migration or cell-cell adhesion between cells grown on NCPs and as 2D monolayers on day 1, showing that cells on NCPs migrated and aggregated without any changes in gene expression (3D Day1 vs 2D Day1 2Fold change.xls). We next characterized mature 3D MCSs on NanoCulture plates, by comparing the gene expression profile of MCSs on day 7 and day 1 (3D Day7 vs 3D Day 1 2Fold change.xls). On day 7, we found a statistically significant increase in the expression of genes related to multicellular organization, intercellular signalling and the response to hypoxia. DNA microarray analysis also showed that the expression of genes that have been reported to be target genes for the transcription factor hypoxia-inducible factor 1 (HIF-1) (Ke, Q. & Costa, M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70, 1469-1480 (2006); Semenza, G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29, 625-634.) was increased on day 7. When compared day 7 with 2D culture cells, the same tendency was observed (3D Day7 vs 2D Day 7 2Fold change.xls).

ORGANISM(S): Homo sapiens

PROVIDER: GSE23773 | GEO | 2010/08/25

SECONDARY ACCESSION(S): PRJNA130831

REPOSITORIES: GEO

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Project description:To characterize cells cultured in 3D culture system using NanoCulture plates (NCPs) (Scivax) and in 2D culture, we examined gene expression in HT-29 cells on day 1 or day 7 on NCPs or 2D monolayer culture, using DNA microarrays. From our study, tumour cells grown on NCPs were morphologically different from cells in 2D monolayers, and the cells on NCPs actively migrated and aggregated to form multicellular spheroids (MCSs). To characterize this cell migration and intercellular adhesion, we compared gene expression in HT-29 cells on day 1 on NCPs and 2D monolayer culture using DNA microarrays. We could not find any significant difference in gene expression related to cell migration or cell-cell adhesion between cells grown on NCPs and as 2D monolayers on day 1, showing that cells on NCPs migrated and aggregated without any changes in gene expression (3D Day1 vs 2D Day1 2Fold change.xls). We next characterized mature 3D MCSs on NanoCulture plates, by comparing the gene expression profile of MCSs on day 7 and day 1 (3D Day7 vs 3D Day 1 2Fold change.xls). On day 7, we found a statistically significant increase in the expression of genes related to multicellular organization, intercellular signalling and the response to hypoxia. DNA microarray analysis also showed that the expression of genes that have been reported to be target genes for the transcription factor hypoxia-inducible factor 1 (HIF-1) (Ke, Q. & Costa, M. Hypoxia-inducible factor-1 (HIF-1). Mol Pharmacol 70, 1469-1480 (2006); Semenza, G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29, 625-634.) was increased on day 7. When compared day 7 with 2D culture cells, the same tendency was observed (3D Day7 vs 2D Day 7 2Fold change.xls). HT-29 cells were maintained routinely as 2D monolayers in a humidified atmosphere of 5% CO2 in air at 37 °C in the growth medium recommended by suppliers. Exponentially growing cells were used in experiments, after trypsinization to detach them from the plates. Viable cells were counted using trypan blue dye-exclusion. For experiments, cells were seeded in 96-well plates at 1×10^4 cells/100 ul of NanoCulture Medium-M (Scivax) in either NCPs (Scivax), or in polystyrene plates for 2D monolayers, for 1 day or 7 days. Cells were collected and used for RNA extraction and DNA microarray analysis.

Project description:The liver is the most common site for colorectal cancer (CRC) metastasis and new tissue culture models are needed to study hepatic metastasis from CRC as none of the current models mimic the biological, biochemical and structural characteristics of the metastatic microenvironment. Decellularization provides a novel approach for the study of cancer extracellular matrix (ECM) as decellularized scaffolds retained their tissue-specific features and biological properties. In the present study we created a 3D model of CRC and matched CRC liver metastasis (CRLM) using patient-derived decellularized ECM scaffolds seeded with HT-29 CRC cell line. Here we show increased HT-29 cell proliferation and migration capability when cultured in cancer-derived scaffolds compared to same-patient healthy colon and liver tissues. CRLM scaffolds also induced activation of epithelial-mesenchymal transition (EMT) with cancer cells showing loss of E-cadherin expression and increased Vimentin expression. EMT was confirmed by gene expression profiling, with the most represented biological processes in CRLM-seeded scaffolds involving demethylation, deacethylation, cellular response to stress metabolic processes, response to oxygen level and to starvation. The complex 3D culture environment reduced the HT-29 cell response to anti-CRC drug 5-FU when used at standard IC50 determined in 2D cultures. In conclusion, our 3D culture system with patient-derived tissue-specific decellularized ECM better recapitulates the metastatic microenvironment compared to conventional 2D culture conditions and represents a relevant approach for the study of liver CRC metastasis formation and progression. Transcriptomic analysis was performed comparing the global gene expression profiles of HT29 cells grown on CRLM and HL scaffolds. The transcriptomic profile of each 3D subtype was compared with HT29 cells grown in plastic. Hierarchical clustering analysis revealed that the gene expression signature of recellularized CRLM scaffolds was more comparable to recellularized HL scaffolds, than to HT29 cells grown in 2D. Gene set enrichment analysis (GSEA) of the differently expressed genes (DEG) up-regulated in recellularized CRLM scaffolds compared to HT29 grown in 2D revealed that genes involved in demethylation, deacethylation and metabolic process belong to the most enriched biological pathways in repopulated-CRLM scaffolds. Comparing the DEG between recellularized CRLM scaffolds vs HT29 grown in 2D with a series of a-priori defined gene-set, called Hallmark, we obtained concordant results with the “HYPOXIA” and “EPITHELIAL_MESENCHYMAL_TRANSITION” pathway, enriched in recellularized CRLM, supporting the idea that 3D culture models are the ability to re-create a complex environment in terms of oxygen tension, nutrients, and metabolic gradient, similarly to the in vivo environment.

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Characterization of HT-29 cells cultured in NanoCulture plates

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