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

Figure 2.

Figure 2

NOTUM has potent tumour suppressive activity in normal and APC-mutant mouse colon tumouroids. (A) Clonal seeding efficiency of wild-type (WT) mouse colon organoids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) (n=3 technical replicates). (B) Images of WT mouse organoids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) 4 days after seeding (n=3 technical replicates). Scale bar: 100 µm. (C) QRT-PCR analysis of canonical Wnt/B-CATENIN pathway target genes including Axin2, Ascl2, Ccnd1 and Lgr5 in organoids from (B). (D) MTT proliferation assays of WT organoids infected with empty lentiviral vector (EV) or Notum overexpression vector (OE) (n=3 technical replicates). (E) Relative expression level of Wnt pathway target genes including Axin2, Ascl2, Ccnd1 and Lgr5 in WT organoids infected with EV or Notum OE (n=3 technical replicates, with one represent). (F) Images of Apc mutant mouse tumouroids infected with EV or vector overexpressing Notum 4 days after seeding (n=3 technical replicates, with one representative of three independent experiments shown). Scale bar: 200 µm. (G) Seeding efficiency of Apc mutant tumouroids from (F). (H) MTT proliferation assays of Apc mutant tumouroids from (F) (n=3 technical replicates, with one representative of three independent experiments shown). (I) Images of Apc mutant mouse tumouroid cultures infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) 4 days after seeding (n=3 biological replicates). Scale bar: 200 µm. (J) The percentage of cells in S-phase (EdU+) in cultures from (I). (K) Clonal seeding efficiency of cells from tumouroids in (I). (L) Percent lumen occlusion was measured after orthotopic implantation of Apc-mutant tumouroids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) (n=4 biological replicates). (M) Weight of primary, orthotopic Apc mutant mouse colon tumours with or without Notum loss of function (n=4 biological replicates). (N) The number of macroscopically observable liver metastases in mice harbouring orthotopic from (L, M). For all panels: *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. See (online supplemental figure 8,9) for validation of driver mutations, sgRNA knockdown and overexpression.