Volume 138, Issue 4, Pages 1374-1383 (April 2010) Phosphatidylinositol 3-Kinase γ Inhibition Ameliorates Inflammation and Tumor Growth in a Model of Colitis-Associated Cancer  Ana González–García, Jesús Sánchez–Ruiz, Juana M. Flores, Ana C. Carrera  Gastroenterology  Volume 138, Issue 4, Pages 1374-1383 (April 2010) DOI: 10.1053/j.gastro.2009.12.001 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Reduced inflammation in PI3Kγ−/− mice. (A) Survival percentage of WT and PI3Kγ−/− mice after 5 DSS treatment rounds. *P < .05, Mantel–Cox test (n = 30). (B) Differences in body weight at the end of treatment (n = 8). Statistical differences were determined using a 2-tailed Student t test. *P < .05 (compared with WT mice). (C) Inflammation score in colons from WT, PI3Kγ+/−, and PI3Kγ−/− mice at the end of azoxymethane (AOM)/DSS treatment (mean ± SEM for 3 independent experiments; n = 10). Statistical differences were determined using a 2-tailed Student t test. *P < .001, **P < .0001 (compared with WT mice). (D) Representative H&E-stained colon sections showing areas of severe inflammatory reaction in WT mice (black arrowheads) and few inflammatory cells in PI3Kγ−/− mice (white arrowheads). Asterisks indicate ulcerated epithelium. Original magnification, 20×; insets were enlarged 3.5×. (E) H&E-stained colon sections showing transmural inflammation in WT mice and superficial inflammation in PI3Kγ−/− mice. Original magnification, 20×. Gastroenterology 2010 138, 1374-1383DOI: (10.1053/j.gastro.2009.12.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 PI3Kγ−/− mice are resistant to inflammation-associated cancer. (A) Tumor incidence in WT, PI3Kγ+/−, and PI3Kγ−/− mice (n = 25). The difference in tumor incidence between WT and PI3Kγ−/− groups is significant (P < .0005, Fisher exact test). (B) Tumor multiplicity in WT, PI3Kγ+/−, and PI3Kγ−/− mice (mean ± SEM, n = 25). Statistical differences were determined using a 2-tailed Student t test. *P < .0001. (C) Distribution of tumor size in WT, PI3Kγ+/−, and PI3Kγ−/− mice (P < .05, χ2 test). (D) Representative images of colons from WT and PI3Kγ−/− mice showing tumors in the distal region. Scale bar, 1 mm. (E) H&E-stained colon sections showing tumors in WT and PI3Kγ−/− mice. Original magnification, 2×. (F) Distribution of tumor types in WT and PI3Kγ−/− mice. Mice were assigned to a category when ≥60% of their tumors were of this type. *P < .05, χ2 test. Gastroenterology 2010 138, 1374-1383DOI: (10.1053/j.gastro.2009.12.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Acute colon inflammation in PI3Kγ−/− mice. (A) Disease activity index in WT and PI3Kγ−/− mice after DSS treatment (mean ± SEM, n = 10); *P < .05, 2-way ANOVA test. (B) Inflammation scores on day 3, 6, or 14 after termination of DSS treatment. *P < .05, 2-tailed Student t test. (C) Number of proliferating (bromodeoxyuridine positive cells) cells per crypt after acute inflammation (mean ± SEM, n = 3). (D) Number of apoptotic (active caspase 3-positive cells) and proliferating (Ki67+ cells) cells per crypt after AOM/DSS treatment (mean ± SEM, n = 4). At least 120 crypts were counted per sample (C and D). Gastroenterology 2010 138, 1374-1383DOI: (10.1053/j.gastro.2009.12.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Impaired innate immune response to DSS in PI3Kγ−/− mice. (A) Numbers of Gr1+ and F4/80+ cells in lamina propria. Statistical differences were determined using a 2-tailed Student t test. *P < .005. (B) Cytokine levels in whole colon cultures. Proinflammatory cytokines were measured by multiplex analysis. A 2-tailed Student t test was used for statistical analysis, *P < .05, **P < .005; mean ± SEM, n ≥ 8 (A and B). Gastroenterology 2010 138, 1374-1383DOI: (10.1053/j.gastro.2009.12.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Altered immune response to DSS in PI3Kγ−/− mice. (A) Total number of B220+ and CD3+ cells in lamina propria. (B) Total number of CD4+ and CD8+ cells in the intraepithelial compartment (left) and lamina propria (right). (C) T helper cell 1 and T helper cell 2 cytokine levels in whole colon cultures measured by multiplex analysis. Statistical differences were determined using a 2-tailed Student t test. *P < .05. Mean ± SEM, n ≥ 8 (A–C). Gastroenterology 2010 138, 1374-1383DOI: (10.1053/j.gastro.2009.12.001) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 γδ and dendritic cell populations in PI3Kγ−/− mice. (A) Percentage (left) and absolute number (right) of γδ T cells in the intraepithelial compartment (mean ± SEM, n ≥ 8). (B) Percentage of dendritic cells in lamina propia (mean ± SEM, n = 5). (C) Percentage of CD4+ cells in the intraepithelial compartment after bone marrow transplant (mean ± SEM, n ≥ 5). Statistical differences were determined using a 2-tailed Student t test. *P < .05. Gastroenterology 2010 138, 1374-1383DOI: (10.1053/j.gastro.2009.12.001) Copyright © 2010 AGA Institute Terms and Conditions