Figure 3.

H2R deficiency promoted the development of inflammation-associated colon cancer in Apc^min/+mice. (A) Representative images taken during colonoscopy of mice at the end of the second recovery phase. (B and C) Tumor numbers were counted and measured in the entire colon of Apc^min/+ (n = 10) and Apc^min/+; H2R KO (n = 9) mice under a dissecting microscope. (D) Infiltrating neutrophils stained with S100A8 antibody (green) (magnification: 40×, 200×, and 1000×). (E) The mean numbers of neutrophils in 8 fields at 200× power for each Apc^min/+ (n = 4) and each Apc^min/+; H2R KO (n = 4) mouse. (F) Immunofluorescence of S100A8 (green) and PD-L1 (red) for colon tumors (magnification: 40×, 200×, and 400×). The red arrowhead in the top panel indicates the Apc^min/+ tumor cells that do not express PD-L1, and the red arrow in the bottom panel indicates the tumor cells that express PD-L1 in Apc^min/+; H2R KO mice. (G) Incidence of PD-L1–positive tumors for Apc^min/+ (n = 8) and Apc^min/+; H2R KO (n = 8) mice. (H) Immunofluorescence (magnification: 40× and 200×) of S100A8 (green) and PD-L1 (red) for Apc^min/+; H2R KO tumors. (I) Immunofluorescence (magnification: 40× and 200×) of proliferating cell nuclear antigen (PCNA) (red) and smooth muscle actin (SMA) (green). The arrow indicates invading tumor cells through the muscularis mucosae into the submucosa. (J) Percentage of invasion in tumors of Apc^min/+ (n = 9) and Apc^min/+; H2R KO (n = 9) mice. In all panels, means ± SD are shown. ^∗∗P < .01 and ^∗∗∗P < .001. DAPI, 4′,6-diamidino-2-phenylindole.