Prospective derivation of a 'Living Organoid Biobank' of colorectal cancer patients
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE65253 | GEO | 2015/05/07
SECONDARY ACCESSION(S): PRJNA273501
REPOSITORIES: GEO
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Project description:In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal (CRC) patients. For most, organoids were also generated from adjacent normal tissue. The organoids closely resemble the original tumor. The spectrum of genetic changes observed within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to robotized, high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43 (rather than in APC). Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. We generated organoids from healthy tissue and coloncarcinoma tissue. The organoids were trypsinized, plated in matrigel and overlaid with medium. After three days, RNA was isolated using Qiagen RNAeasy. Medium conditions are the same for all organoids, irrespective of their origin.
2015-05-07 | E-GEOD-64392 | biostudies-arrayexpress
Project description:In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal (CRC) patients. For most, organoids were also generated from adjacent normal tissue. The organoids closely resemble the original tumor. The spectrum of genetic changes observed within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to robotized, high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43 (rather than in APC). Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design.
2015-05-07 | GSE64392 | GEO
Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.
2015-05-07 | E-GEOD-65253 | biostudies-arrayexpress
Project description:Patient derived organoids (PDOs) have been established as a 3D culture model which closely recapitulates the in vivo tumor biology. However, one limitation of this culture model is the lack of tumor microenvironment which has a significant role in tumor progression and drug response. To address this, we established and molecularly characterized a novel 3D co-culture model of colorectal cancer (CRC) based on PDOs and patient matched fibroblasts. Both normal and cancer associated fibroblasts, NFs and CAFs respectively, were able to support organoid growth without addition of niche factors to the media. Additionally, co-cultures showed closer resemblance to primary patient material than organoid mono-cultures as evaluated by histology. Finally, RNA gene expression signatures of tumor cells and fibroblasts isolated from mono- or co-cultures demonstrated that co-cultures support greater cell type heterogeneity. In this proteomics dataset we compared pairs of NFs and CAFs derived from five patients. Collectively, we present a newly established human derived organoid-fibroblast model which, closely recapitulates in vivo tumor heterogeneity and involves the tumor microenvironment.
2023-03-14 | PXD032047 | Pride
Project description:The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Loss of SMAD4 is a key event in late-stage CRC resulting in the progression to metastatic CRC in 10-30% of the cases. However, the biological processes and underlying molecular mechanisms that it affects are not fully understood. Here, we applied a multi-omics approach to a CRC tumor progression organoid model that faithfully reflects the metastasis-inducing effects of SMAD4 inactivation. We show that loss of SMAD4 results in loss of differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFB, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by metastatic organoids – DKK3 – reduces the anti-tumor effects of natural killer cells (NKCs). Altogether, our data provides promising new targets concerning the role of SMAD4 perturbation and metastatic disease in CRC.
2022-12-11 | PXD036441 | Pride
Project description:Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signaling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by Partial Duct Ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) which expand 5-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality We generated arrays from whole pancreas, islet, acinar and duct (Sox9+ sorted) cells and pancreas derived cultures maintained in our defined medium. For the clustering analysis we substracted the pancreas array to all arrays. Genes 2 fold differentially expressed in the ductal array were used for the analsyis. For the genes enriched in organoid cultures compared to pancreas we substracted the organoid culture arrays to the pancreas array. Genes >2-fold differentially expressed were used for the analysis.
2013-09-17 | E-GEOD-50103 | biostudies-arrayexpress
Project description:WNT and/or Rspondin was withdrawn from the medium and Wnt pathway inhibitors IWP2 and DKK1 were added to the growth medium of human stomach, liver, pancreas, small intestinal and colon organoids and RNA was harvested at time points 0h, 6h and 24h to identify Wnt regulated genes.
2021-02-22 | GSE167115 | GEO
Project description:The unfolded protein response maintains endoplasmic reticulum (ER) homeostasis by sensing protein-folding stress and orchestrating cellular adaptation via the ER-transmembrane proteins IRE1, PERK and ATF6. Malignant cells can co-opt IRE1 and PERK to sustain growth; however, the importance of ATF6 in cancer remains poorly deciphered. We observed elevated ATF6 transcriptional activity in several cancers including colorectal carcinoma (CRC). Genetic silencing or small molecule inhibition of ATF6 blocked cell cycle progression and reduced viability of several human CRC cell lines in vitro and disrupted tumor progression in vivo. Unexpectedly, ATF6 interference disabled Wnt and Myc signaling and reduced stemness. ATF6 inhibition attenuated growth of organoids derived from malignant but not normal human intestinal tissue, decreasing Wnt-pathway activity and driving cellular differentiation. Wnt-surrogate agonism in a Wnt-dependent CRC organoid restored pathway activity and rescued growth under ATF6 blockade. Our findings identify ATF6 as an unexpected facilitator of oncogenic Wnt signaling in CRC.
2024-08-06 | GSE273988 | GEO
Project description:The unfolded protein response maintains endoplasmic reticulum (ER) homeostasis by sensing protein-folding stress and orchestrating cellular adaptation via the ER-transmembrane proteins IRE1, PERK and ATF6. Malignant cells can co-opt IRE1 and PERK to sustain growth; however, the importance of ATF6 in cancer remains poorly deciphered. We observed elevated ATF6 transcriptional activity in several cancers including colorectal carcinoma (CRC). Genetic silencing or small molecule inhibition of ATF6 blocked cell cycle progression and reduced viability of several human CRC cell lines in vitro and disrupted tumor progression in vivo. Unexpectedly, ATF6 interference disabled Wnt and Myc signaling and reduced stemness. ATF6 inhibition attenuated growth of organoids derived from malignant but not normal human intestinal tissue, decreasing Wnt-pathway activity and driving cellular differentiation. Wnt-surrogate agonism in a Wnt-dependent CRC organoid restored pathway activity and rescued growth under ATF6 blockade. Our findings identify ATF6 as an unexpected facilitator of oncogenic Wnt signaling in CRC.
2024-08-08 | GSE274172 | GEO
Project description:We previously established long-term 3D organoid culture systems for several murine tissues (intestine, stomach, pancreas and liver) as well as human intestine and pancreas. Here, we describe culture conditions to generate long-term 3D culture from human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Human gastric cultures can expand indefinitely in 3D Matrigel. Cultures can be generated from normal tissue, from single sorted stem cells, or from tumor tissue. Organoids maintain many characteristics of the respective tissue in terms of histology, marker expression and euploidy. Organoids from normal tissue express markers of four lineages of the stomach and self-organize in gland and pit-domains. They can be directed to specifically express either lineages of the gastric gland, or the gastric pit by addition of Nicotinamide and withdrawal of Wnt. While gastric pit lineages react marginally to bacterial infection, gastric gland lineages mount a strong inflammatory response. The gastric culture system provides a unique tool to study gastric pathologies. We generated 2 sets of experiments. The first set contains organoids in 4 conditions: (1) organoids in expansion condition ENRWFGNiTi ("gland-type organoids") from 3 donors, (2) organoids as in 1 but differentiated for 4 days in differentiation condition ENR_FGNiTi ("pit'type organoids"), (3) organoids as in 1 but infected with Helicobacter pylori strain P12 MOI 50 for 2 h, (4) organoids as in 2 but infected as in 3. All 4 conditions were tested on the same organoid line in parallel. This experiment was conducted independently with cultures from 3 different donors. The second set of experiments compares freshly isolated glands with organoids. Samples from 2 patients were analyzed. Each patient received a total gastrectomy. From each patient, glands from corpus region or pyloric antrum were isolated. From each isolation, one aliquod was stored for microarray analysis and one aliquod used to generate organoids. Organoids and glands were subsequently lysed and analyzed in parallel.
2014-10-13 | E-GEOD-60557 | biostudies-arrayexpress