Dataset Information

Bioreactor-engineered cancer tissues mimic phenotypes, gene expression profiles and drug resistance mechanisms detectable in xenografts and clinical specimens.

ABSTRACT: Cancer tissue-like structures were developed by using established human tumor cell lines in perfusion-based bioreactor systems. In colorectal cancer (CRC) cell lines, perfusion allowed more homogeneous scaffold seeding than tri-dimensional (3D) static cultures and significantly (13.7 fold, p<0.0001) higher proliferation. Resulting tissues exhibited morphology and phenotypes similar to xenografts generated in immunodeficient mice. Whole transcriptome analysis of 2D, 3D static and 3D perfusion cultures revealed the highest correlation between xenografts and 3D perfusion cultures (r=0.985). Clinically relevant concentrations of 5-FU, used in neo- and adjuvant CRC treatment, had no effect on numbers of HT-29 CRC cells cultured in 3D perfusion or xenografts, as compared with a 55.8% reduction in 2D cultures. Treatment induced apoptosis in 2D cultures, but only “nucleolar stress” in perfused cells and xenografts, consistent with partial responsiveness. In 3D perfusion cultures BCL-2, TRAF1, and FLIP gene expression was marginally affected, as compared with significant down-regulation in 2D cell cultures. Accordingly, ABT-199 BCL-2 inhibitor, induced cytostatic effects in 3D perfusion but not in 2D cell cultures (p=0.003). Tumor cells from partially responsive (Dworak 2) patients undergoing neo-adjuvant treatment, typically (10/11) expressed BCL-2, as compared with 0/3 highly (Dworak 3-4) responsive and 4/15 fully resistant CRC (Dworak 0/1, p=0.03), closely matching 3D perfusion cultures data. These results indicate that 3D perfusion cultures efficiently mimic phenotypic and functional features observed in xenografts and clinical specimens. These models may be of critical translational relevance to address fundamental human tumor cell biology issues and to develop predictive pre-clinical tests of novel compounds.

ORGANISM(S): Homo sapiens

PROVIDER: GSE57961 | GEO | 2016/01/25

SECONDARY ACCESSION(S): PRJNA248557

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Development of systems allowing the maintenance of native properties of mesenchymal stromal cells (MSC) is a critical challenge for studying physiological functions of skeletal progenitors, as well as towards cellular therapy and regenerative medicine applications. Conventional stem cell culture in monolayer on plastic dishes (2D) is associated with progressive loss of functionality, likely due to the absence of a biomimetic microenvironment and the selection of adherent populations. Here we demonstrate that 2D MSC expansion can be entirely bypassed by culturing freshly isolated bone marrow cells within the pores of 3D scaffolds in a perfusion-based bioreactor system, followed by enzymatic digestion for cell retrieval. The 3D-perfusion system supported MSC growth while maintaining cells of the hematopoietic lineage, and thus generated a cellular environment mimicking some features of the bone marrow stroma. As compared to 2D-expansion, sorted CD45- cells derived from 3D-perfusion culture after the same time (3 weeks) or a similar extent of proliferation (7-8 doublings) maintained a 4.3-fold higher clonogenicity and exhibited a superior differentiation capacity towards all typical mesenchymal lineages, with similar immunomodulatory function in vitro. Transcriptomic analysis performed on MSC from 5 donors validated the robustness of the process and indicated a reduced inter-donor variability as well as a significant upregulation of multipotency-related gene clusters following 3D-perfusion as compared to 2D expansion. The described system offers a model to study how factors of a 3D engineered niche may regulate MSC function and, by streamlining conventional labor-intensive processes, is prone to automation and scalability within closed bioreactor systems. Nucleated cells were isolated from 5 fresh human bone marrow aspirates by means of red blood cells lyses buffer and then were seeded into a 3D perfusion bioreactor system using a pure hydroxyapatite 3D scaffold and in conventional Petri dishes (2D). After culture for 19 days, cells from both systems were enzymatically retrieved and sorted using anti-CD45-coated magnetic beads. Total RNA was extracted from CD45- cells, QCed and hybridized to Affymetrix microarrays.

Project description:The development of more complex but reliable systems for compound testing in a pharmaceutical context is a challenging task to date. Three-dimensional (3D), organ mimetic cell culture is aiming to become an alternative to common two-dimensional (2D) cell culture or animal testing in that field. We developed a biocompatible 3D cell culture environment for a hepatocellular carcinoma (HCC) model that enables cellular maintenance in a polycarbonate scaffold structure. Albumin, regarded as a differentiation marker, was elevated in statically 3D cultivated HepG2 cells. Expression of HCC tumor marker alpha-fetoprotein (AFP) was reduced compared to immunofluorescence stainings of 2D cultivated cells. Remarkably, expression of cytokeratin and pathophysiologically relevant beta-1 integrin (ITGB1) was found enhanced in nonperfused 3D cell culture. Changes in gene expression induced by the 3D cultivation environment were investigated using Ingenuity Pathway Analysis (IPA). Our findings revealed involvement of the insulin growth factor (IGF) signaling pathway in upregulation of matrix metalloproteinases (MMP) and ITGB1. The experimental data indicate a more differentiated state in 3D cultivated HepG2 cells than in the respective 2D experiments. Hence, scaffold-supported 3D cultivation of HepG2 cells may lead to a gain of information valuable for both drug testing and cancer research. HepG2 cells were cultivated for five days under 2D and 3D statical and perfused conditions. Cultivation was started with 0.25x10^6 cells in 2D and with 1x10^6 vital cells for the 3D experiments. The day of seeding was defined as d0. The groups were classified as follows: 2D, i.e., monolayer cultures, 3D, i.e., statical 3D cultures and BR, which denotes perfused 3D culture of HepG2 cells. The perfusable bioreactor system was operated using a peristaltic pump. It houses the MatriGrid, a polycarbonate-based microporous cellular support. For 3D static cultivation, cell-inoculated MatriGrids were placed in wells of a 24-well plate. Microarray experiments of three 2D (i.e., control), three 3D statically and three actively perfused 3D cultivations were performed at SIRS-Lab GmbH (SIRS-Lab GmbH, Jena, Germany) according to the manufacturer's instructions (Illumina, San Diego, CA). Altogether, 9 RNA samples of hepatocyte cultures and an internal control RNA were hybridized on two HumanHT-12 v4 Expression BeadChips.

Project description:The traditional method for studying cancer in vitro is to grow immortalized cancer cells in two-dimensional (2D) monolayers on plastic. However, many cellular features are impaired in these unnatural conditions and big alterations in gene expression in comparison to tumors have been reported. Three-dimensional (3D) cell culture models have become increasingly popular and are suggested to be better models than 2D monolayers due to improved cell-to-cell contacts and structures that resemble in vivo architecture. The aim of this study was to develop a simple high-throughput 3D drug screening method and to compare drug responses in JIMT1 breast cancer cells when grown in 2D, in polyHEMA coated anchorage independent 3D models and in Matrigel on-top 3D cell culture models. We screened 102 compounds with multiple concentrations and biological replicates for their effects on cell proliferation. The cells were either treated immediately upon plating or they were allowed to grow in 3D for four days prior to the drug treatment. Big variations in drug responses were observed between the models indicating that comparisons of culture model influenced drug sensitivities cannot be made based on effects of a single drug. However, we show with the 63 most prominent drugs that, in general, JIMT1 cells grown on Matrigel were significantly more sensitive to drugs than cells grown in 2D cultures, while responses of cells grown in polyHEMA resembled those of 2D. Furthermore, comparison of gene expression profiles of the cell culture models to xenograft tumors indicated that cells cultured in Matrigel and as xenografts most closely resembled each other. In this study we also suggest that 3D cultures can provide a platform for systematic experimentation of larger compound collections in a high-throughput mode and be used as alternatives for traditional 2D screens towards better comparability to in vivo state. Gene expression analysis of JIMT1 breast cancer cells cultured as xenografts for 43 days, in two dimensional cultures for seven days (2D7d), in polyHEMA three dimensional cell culture models for four and seven days (PH7d and PH7d), and in Matrigel three dimensional cultures for four and seven days (MG4d and MG7d). Two biological replicates was included for each sample.

Dataset Information

Bioreactor-engineered cancer tissues mimic phenotypes, gene expression profiles and drug resistance mechanisms detectable in xenografts and clinical specimens.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure