Project description:Using 5' droplet-based single cell sequencing, we profiled single cells dervied from human colorectal cancer organoids carrying either APC mutation or RSPO fusion, and paired normal colon organoids for the later.
Project description:This study has two components: (1) Human colon adenoma organoids (n=4 patients) were dissociated into single cells. Cells were incubated with a magnetic bead bound to an LGR5 antibody and run through a magnetic column. Magnet bound cells and flow through negative (FTN) cells were obtained. Magnet bound and FTN cells were incubated with an APC-check reagent (which binds to the magnetic bead on the LGR5 antibody) and DAPI, before being sorted by flow cytometry. 3 populations of live (DAPI-) cells were collected: FTN: Flow through negative. LGR5 negative by magnet and by flow cytometry SortedNeg: Magnet bound cells that were negative for LGR5 by flow cytometry SortedPos: Magnet bound cells that were positive for LGR5 by flow cytometry (2) Human colon organoids, as well as the tissue the organoid was derived from and adjcacent normal tissue (from n=19) were also profiled for known colorectal cancer associated mutations using the Qiagen Qiaseq Colorectal Cancer Panel, which provides targeted sequencing information for 71 genes.
Project description:As metabolic rewiring is crucial for cancer cell proliferation, metabolic phenotyping of patient-derived organoids is desirable to identify drug-induced changes and trace metabolic vulnerabilities of tumor subtypes. We established a novel protocol for metabolomic and lipidomic profiling of colorectal cancer organoids by LC-QTOF-MS facing the challenge of capturing metabolic information from minimal sample amount (< 500 cells/injection) in the presence of extracellular matrix (ECM). The best procedure of the tested protocols included ultrasonic metabolite extraction with acetonitrile/methanol/water (2:2:1, v/v/v) without ECM removal. To eliminate ECM-derived background signals, we implemented a data filtering procedure based on p-value and fold change cut-offs which retained features with signal intensities >120% compared to matrix-derived signals present in blank samples. As a proof-of-concept, the method was applied to examine the early metabolic response of colorectal cancer organoids to 5-fluorouracil treatment. Statistical analysis revealed dose-dependent changes in the metabolic profiles of treated organoids including elevated levels of 2'-deoxyuridine, 2'-O-methylcytidin, inosine and 1-methyladenosine and depletion of 2'-deoxyadenosine and specific phospholipids. In accordance with the mechanism of action of 5-fluorouracil, changed metabolites are mainly involved in purine and pyrimidine metabolism. The novel protocol provides a first basis for the assessment of metabolic drug response phenotypes in 3D organoid models.
Project description:Targeted capture of cancer gene panel bait set in single cell derived organoids from colon tissue and colorectal cancer from 1 patient.
Project description:Here, we generated bulk RNA-seq data on colon organoids derived from both healthy and familial adenomatous polyposis patients. We related findings observed within this dataset to differential expression findings from a publicly available colorectal cancer cohort.