WNT activated cells are the origin of regrowth of colorectal cancer organoids after irradiation
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PROVIDER: PRJNA587924 | ENA |
REPOSITORIES: ENA
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Project description:Regeneration capacity of intestinal epithelial cells are highly dependent on the status of stemness. In the homeostasis, only a small subset of stem cells is responsible for the turnover of the intestinal epithelium. However, at the injury, such as caused by radiation, intestinal epithelial cells show substantial plasticity for regeneration. Most of the colorectal cancers (CRCs) are differentiated adenocarcinoma, which maintain the expression of both stemness and differentiation markers, suggesting an existence of a similar regeneration system as in normal cells, although the role of such fluctuation of stemness in regeneration of cancer after irradiation is not well understood. In the present study, we first examined the effect of radiation on growth, stemness and differentiation using organoids derived from differentiated CRC. Both proliferation and stemness markers were once drastically decreased after sub-lethal dose of irradiation, meanwhile differentiation markers remained. After the static phase, regrowth foci consisting of proliferating cells with expression of the stem cell markers reappeared. We applied the foci formation to a radio-sensitivity assay in CRC organoids. Radio-sensitivity, the ability of foci formation, differed among the examined lines, and correlated well with the in vivo radiation sensitivity. The radio-sensitivity decreased after mechanical disruption of the organoids, which we have reported to increase the stemness of CRC organoids in a WNT dependent manner. Pre-treatment of organoids with histone deacetylase inhibitors increased radiation sensitivity, accompanied with suppressed expression of WNT target genes, and WNT inhibitors increased radio-sensitivity, suggesting that not all but some of the cancer cells with high WNT activity at the timing of radiation is the origin of the foci formation. The radiation sensitivity assay using CRC organoids established in the present study would provide a novel platform to evaluate the radiosensitizers against differentiated adenocarcinoma of CRC.
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Project description:In this project, we aimed to examine gene expression changes in intestinal organoids treated with the complement C5a receptor 1 (C5aR1) antagonist PMX205 either with or without irradiation. Intestinal organoids from wild-type C57BL/6 mice were cultured and plated. The following day, the media was supplemented with 5 μg/ml of PMX205 or a vehicle control, and after one hour, organoids were subjected to either 0 Gy or 9 Gy of radiation. Samples were collected after 24 and 48 hours, and RNA was extracted and processed for RNA sequencing.
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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.
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