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. 2016 Jul 1;27(13):1981–1989. doi: 10.1091/mbc.E15-10-0747

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© 2016 Zasadil et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — High CIN does not suppress tumor initiation in the small intestine. (A) Intestinal organoid with a branched, complex morphology typical of organoids retaining expression of wild- type APC. (B) Consistent with the elevated rates of cell death observed in CENP-E^+/−;Apc^Min/+ MEFs and animals, complex organoids from CENP-E^+/−;Apc^Min/+ animals increase in size more slowly than Apc^Min/+ organoids; n = 30 Apc^Min/+ and 40 CENP-E^+/−;Apc^Min/+ organoids. (C) The layer of normal intestinal epithelium (white arrows) surrounding an intestinal tumor (yellow arrowheads) exhibits cell surface β-catenin staining, whereas β-catenin also exhibits nuclear localization in the tumor cells. (D) As in intact intestine, β-catenin localizes predominantly to the cell surface in complex organoids (left). In cystic organoids (right), β-catenin also shows nuclear staining, consistent with Apc loss and constitutive activation of Wnt signaling. (E) Cystic organoids, which are characteristic of Apc loss (Fatehullah et al., 2013; Onuma et al., 2013), show a rounded appearance that is distinct from the complex, branched structure of organoids expressing APC, as shown in A. Cystic organoids from CENP-E^+/−;Apc^Min/− tumor-initiating cells are substantially larger than cystic Apc^Min/− organoids derived from Apc^Min/+ littermates on day 4 after plating. (F) A higher percentage of cells from CENP-E^+/−;Apc^Min/+ than Apc^Min/+ small intestine form cystic organoids characteristic of Apc loss, demonstrating that high CIN does not inhibit tumor initiation in this context; n = 1782 organoids from Apc^Min/+ and 1514 organoids from CENP-E^+/−;Apc^Min/+ animals. (G) Cystic organoids from tumor-initiating cells generated from CENP-E^+/−;Apc^Min/+ normal intestinal epithelium are substantially larger on day 4 after plating than cystic organoids from Apc^Min/+ littermates. (H) In contrast, cystic organoids from mature CENP-E^+/−;Apc^Min/– tumor cells from adenomas of CENP-E^+/−;Apc^Min/+ animals show no such growth advantage, suggesting that tumor-initiating cells are less sensitive than mature tumor cells to high CIN. (I) Growth rates of cystic organoids from tumor-initiating cells for the 10 d after sizes were measured in G, showing that CENP-E^+/−;Apc^Min/- tumor-initiating cells rapidly lose their early growth advantage. *p < 0.05.