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. 2023 Aug 17;72(12):2294–2306. doi: 10.1136/gutjnl-2022-329140

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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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Notum is a cell-autonomous oncogene in colorectal adenocarcinoma. (A) UpSet plot showing common differentially expressed genes (p<0.05, log2FC>1) in tumour cells from mouse models of CRC relative to normal colonic epithelium. APKS: adenocarcinoma resulting from orthotopic implantation of Apc/Trp53/Kras/Smad4 quadruple-mutant tumouroids into syngeneic mouse colon. Azoxymethane/dextran sodium sulfate (AOM/DSS): inflammation-induced colon adenocarcinoma. Apc^Min/+ : colon adenoma induced by spontaneous loss of Apc heterozygosity in the Apc^min/+ mouse model. Apc LOF: colonic epithelium after acute loss-of-function of Apc in an Apc^flox/flox::Villin-CreER mouse 5 days after tamoxifen-induced Apc inactivation. (B) Uniform manifold approximation projections (UMAP) of single cell RNA sequencing (scRNAseq) datasets from mouse tumour models in (A), with epithelial/carcinoma cells expressing Epcam (top). Notum expression (bottom) is restricted to epithelial/carcinoma cells (circled). (C) Human NOTUM expression in the Cancer Genome Atlas (TCGA) colon (COAD) and rectal (READ) adenocarcinoma datasets. (D) Left: UMAP representation the cell populations of human CRC primary tumour and adjacent normal epithelium. Cells are coloured according to their cell types. (n=17). Right: UMAP showing NOTUM expression restricted to human carcinoma cells. (E) Western blot analysis for NOTUM in mouse APKS tumouroids infected with control sgRNA (sgCtl) or Notum sgRNA(sgNotum). β-ACTIN was used as a loading control. (F) Images of mouse APKS tumouroids from (E) 3 days after seeding (n=3 technical replicates, with one representative of three independent experiments shown). Scale bar: 200 µm. (G) Clonal seeding efficiency of single APKS cells from (E/F). (H) MTT proliferation assays of APKS mouse tumouroids (n=3 technical replicates, with one representative of three independent experiments shown). (I) Left: Primary tumours formed 8 weeks after orthotopic injection of APKS tumouroids into the distal colon of syngeneic recipient mice (n=5 in sgCtl group, n=4 in sgNotum group). Right: Weight of primary tumours from the left. (J) Left: Livers of the mice bearing primary APKS tumours from (I). White asterisks show metastatic liver lesions. Right: The number of macrometastases visible in the liver of mice from (I). For all panels: *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. See online supplemental figures 8,9 for validation of driver mutations and sgRNA knockdown. AOM/DSS, azoxymethane/dextran sodium sulfate; CRC, colorectal cancer.