Volume 153, Issue 4, Pages 1040-1053.e4 (October 2017) MET Signaling Mediates Intestinal Crypt-Villus Development, Regeneration, and Adenoma Formation and Is Promoted by Stem Cell CD44 Isoforms  Sander P.J. Joosten, Jurrit Zeilstra, Harmen van Andel, R. Clinton Mijnals, Joost Zaunbrecher, Annet A.M. Duivenvoorden, Marc van de Wetering, Hans Clevers, Marcel Spaargaren, Steven T. Pals  Gastroenterology  Volume 153, Issue 4, Pages 1040-1053.e4 (October 2017) DOI: 10.1053/j.gastro.2017.07.008 Copyright © 2017 AGA Institute Terms and Conditions

Figure 1 HGF/MET signaling can mediate outgrowth of ISCs into mini-gut organoids, substituting EGFR signals. (A) Development of mini-gut organoids from intestinal crypts after 7 days of culture in medium supplemented with either EGF, HGF, EGF + HGF, or control (no growth factor). (B) Relative mini-gut size at day 10 after plating (n = 10; **P < .01). (C) Time kinetics of organoid expansion stimulated in the presence of HGF-containing or control medium. (D, E) Relative expansion of crypts from Lgr5Creert2/Metfl/fl mice stimulated with HGF or EGF in the presence or absence of 4-hydroxytamoxifen (TAM) (n = 10; ***P < .001). (F) Mini-gut formation starting with sorted LGR5+ stem cells from Lgr5-EGFP-ires-creERT2 small intestines in culture medium supplemented with HGF. Crypt-like structures start to form 1 week after plating. Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Figure 2 MET expression is dispensable for normal intestinal homeostasis. (A, B) β-Gal stain on AhCre (A) and Lgr5Creert2 (B) Metfl/fl and Met+/+ control intestines 7 days after Cre-mediated recombination. (C, D) Quantification of Ki67+ cells and Caspase-3+ cells per crypt (n ≥ 3) in AhCre/Metfl/fl and AhCre/Met+/+ control mice. (E) Quantification of intestinal cell populations for Goblet, Enteroendocrine, and Tuft cells (n ≥ 3) in AhCre/Metfl/fl and AhCre/Met+/+ control mice. (F) Number of β-gal+ crypts per 1000 crypts in Lgr5Creert2/Metfl/fl and Lgr5Creert2/Met+/+ littermate control mice at day 5, 10, or 30 after induction of Cre-mediated recombination (n ≥ 3). Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Figure 3 Met deficiency leads to attenuated repair of radiation-induced damage. (A) Cycling crypt cells in AhCre/Metfl/fl vs AhCre/Met+/+ after Cre-mediated recombination of Met. Representative picture of Ki67 expression at 72 hours after 10 Gy radiation. (B) Quantification of cycling crypts in AhCre/Metfl/fl vs AhCre/Met+/+ mice and in Lgr5Creert2/Metfl/fl vs Lgr5Creert2/Met+/+ after Cre-mediated recombination of Met. Number of Ki67+ crypts/mm measured at 72 hours after 10 Gy radiation (n = 5; ***P < .001). (C) Number of β-gal+ crypts per 1000 crypts in Lgr5Creert2/Metfl/fl vs Lgr5Creert2/Met+/+ control littermates at 72 hours after 10 Gy radiation (n = 10; ***P < .001). (D) Number of Ki67+ cells per β-gal–positive or –negative crypt at 72 hours after 10 Gy radiation. Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Figure 4 MET signaling promotes expansion of adenoma organoids. (A) Example of an adenoma spheroid culture derived from ApcMin/+ mice 1 week after reseeding. (B) Relative expansion of ApcMin/+ derived adenoma organoids stimulated with HGF-containing or control medium (no growth factor) (n = 30; **P < .01). (C, D) Relative expansion of tamoxifen induced Lgr5Creert2/Apcfl/fl-derived adenoma organoids stimulated with HGF- or EGF-containing or control medium (no growth factor) (n = 30; ***P < .001). Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Figure 5 Met deletion attenuates adenoma formation in vivo in both Lgr5Creert2 and AhCre mouse models. (A, B) Number of microadenomas and adenomas present in a single Ki67-stained Swiss roll section of Lgr5Creert2/Apcfl/fl/Metfl/fl or Lgr5Creert2/Apcfl/fl/Met+/+ control mice (n ≥ 6; **P < .01). (C) Adenoma size of (B) (***P < .001). (D) Size distribution of adenomas as percentage of adenomas per group (*P < .05; **P < .01). (E, F) Quantification of Ki67+ cells in (micro)adenoma in AhCre/Metfl/fl and AhCre/Met+/+ control mice (n ≥ 6). (G, H) Quantification of Caspase3+ cells in (micro)adenoma in AhCre/Metfl/fl and AhCre/Met+/+ control mice (n ≥ 6; ***P < .001). (I) Number of intestinal adenomas in AhCre/Apcfl/+/Metfl/fl vs AhCre/Apcfl/+/Met+/+ littermates 16 weeks after Cre-induction (n = 14; *P < .05). (J) Number of microadenoma in AhCre/Apcfl/+/Metfl/fl vs AhCre/Apcfl/+/Met+/+ scored on Ki67-stained tissue sections (n = 14; *P < .05). Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Figure 6 Intestinal stem cell CD44v isoforms supports HGF-dependent mini-gut formation and adenoma growth. (A) Relative size of small intestinal organoids from crypts of Cd44+/+, Cd44−/−, Cd44s/s, and Cd44v4–10/v4–10 mice cultured for 10 days in medium supplemented with EGF (50 ng/mL). (B) Organoids from the same batches of crypts as in (A) cultured for 10 days in medium supplemented with HGF (100 ng/mL) (n = 15; **P < .01). (C) Representative pictures of small intestinal organoids from crypts of Cd44+/+, Cd44−/−, Cd44s/s, and Cd44v4–10/v4–10 mice cultured in medium supplemented with HGF (100 ng/mL). (D, E) Expansion kinetics of Cd44s/s and Cd44v4–10/v4–10 adenoma spheroids cultured with either HGF-containing or control medium (n = 15; **P < .01). (F) Quantification of Cd44s/s and Cd44v4–10/v4–10 spheroid size at day 3 after plating in either HGF-containing or control medium (n = 15; **P < .01; ***P < .001). Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 1 HGF/Met signaling can mediate outgrowth of ISCs into “mini-gut” organoids containing all differentiated cell types, substituting Egfr signals. (A) Time kinetics of organoid expansion stimulated with EGF-, HGF-, or EGF + HGF-containing or control medium for 21 days. (B) Percentage of GFP-expressing organoids after 10 days of culturing in the presence of tamoxifen (n ≥ 50 per condition; **P < .01). (C) (Immuno)histochemical analysis of HGF-stimulated organoids for differentiation markers Lysozyme (Paneth cells), Pas-D (Goblet cells), Synapthofysin (Enteroendocrine cells), Dclk1 (Tuft cells), and Alkaline Phosphatase (differentiated cells). Arrows indicate positively stained cells. Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 2 Efficient recombination of the Metfl allele. (A) Percentage recombined DNA per tissue section in AhCre or Lgr5Creert2 x Metfl/fl or Metfl/+. Quantitative polymerase chain reaction analysis using primers flanking and overlapping the recombination site (n = 3). (B) Adenomas derived from Apcfl/+ mice have recombined Metfl alleles. Polymerase chain reaction for presence of 3′ LoxP site and recombination polymerase chain reaction-product MetΔ. Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 3 (A, B) Quantification of Ki67+ proliferating cells and Caspase-3+ apoptotic cells per crypt per intestinal section in AhCre/Metfl/fl and AhCre/Met+/+ control mice (n ≥ 3 per condition; Ki67 counted unilateral). (C) (Immuno) histochemical identification of intestinal cell populations. Goblet cells (Alcian blue); Enteroendocrine cells (Synapthofysin); Tuft cells (Dclk1), and Enterocytes (Alkaline Phosphatase) in AhCre/Metfl/fl or AhCre/Met+/+ control mice 4 weeks after induction of recombination. Arrows indicate enteroendocrine cells. Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 4 (A, B) (Micro)-Adenoma distribution of LgrCreert2/Apcfl/fl/Metfl/fl and LgrCreert2/Apcfl/fl/Met+/+ control mice (n ≥ 6 per condition; *P < .05; **P < .01). (C) Adenoma distribution of AhCre/Apcfl/+/Metfl/fl and AhCre/Apcfl/+/Met+/+ control mice (n = 14 per condition; **P < .01). (D) Relative adenoma size as percentage of total adenoma per group in AhCre/Apcfl/+/Metfl/fl and AhCre/Apcfl/+/Met+/+ control mice (n = 14 per condition). Gastroenterology 2017 153, 1040-1053.e4DOI: (10.1053/j.gastro.2017.07.008) Copyright © 2017 AGA Institute Terms and Conditions