Volume 140, Issue 1, Pages 310-321.e4 (January 2011) CXCL17 and ICAM2 Are Associated With a Potential Anti-Tumor Immune Response in Early Intraepithelial Stages of Human Pancreatic Carcinogenesis  Nobuyoshi Hiraoka, Rie Yamazaki–Itoh, Yoshinori Ino, Yasunori Mizuguchi, Tesshi Yamada, Setsuo Hirohashi, Yae Kanai  Gastroenterology  Volume 140, Issue 1, Pages 310-321.e4 (January 2011) DOI: 10.1053/j.gastro.2010.10.009 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 (A−D) Immunohistochemical detection of CD207+ DCs and CD8+ T cells infiltrating into IPMA and IPMC. Low-power view (A), medium-power view (D), and high-power view (B, C). B corresponds to the rectangle in A. Two infiltration patterns of T cells and DCs were found, which were intraepithelial infiltration (arrows) and infiltration into the stroma (clear arrowheads). IPMA is on the right side of the line, and IPMC on the left (B). (E, F) Immunohistochemical detection of the maturation state of DCs in regional lymph nodes (LNs). Representative feature of a regional LN from a patient with IPMA with immunohistochemistry for (E) CD207 and (F) CD208. Gastroenterology 2011 140, 310-321.e4DOI: (10.1053/j.gastro.2010.10.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 (A, B) Absolute number of intraepithelial infiltrating CD1a+ or CD207+ myeloid DCs (A, left) and CD8+ T cells (B), and the ratio of intraepithelial DCs among total tumor-infiltrating DCs (A, right). (C) Absolute number of CD207+ immature DCs (left) and mature CD208+ DCs (center), and the ratio of mature CD208+ DCs to CD207+ immature DCs in regional lymph nodes (right). Significant value of *P < .05; **P < .005; ***P < .001 in (A, B), and *P < .05 and **P < .01 in (C). Gastroenterology 2011 140, 310-321.e4DOI: (10.1053/j.gastro.2010.10.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 (A) Expression of chemokine genes that can induce migration of monocytes or DCs during pancreatic carcinogenesis determined by real-time reverse transcribed polymerase chain reaction. Bars represent mean ± standard deviation. Significant value of *P < .05; **P < .01; and ***P < .001. Expression of the CCL25 gene was not detected in any of them (data not shown). (B) Western blot analysis revealed that chemokine (C-X-C motif) ligand 17 (CXCL17) (14 kD) was expressed in 70% (7 of 10) cases of IPMA, in 10% (1 of 10) cases of IPMC, in 13% (4 of 30) cases of pancreatic ductal adenocarcinoma, and none (0 of 10) of normal pancreas tissue. Results in the Western blot analysis are representative cases. Summarized results shown in the lower column. (C) In vitro chemotaxis assays revealed that CXCL17 induced the migration of CD14+ monocytes (left) but not CD3+ T cells (right), CD19+ B cells, or CD56+ natural killer cells (data not shown). Data represent 1 of 5 independent experiments. (D) CXCL17 induced the migration of monocyte-derived DCs regardless of their maturation state, but did not induce the migration of monocyte-derived Langerhans cells (LCs). Data represent 1 of 3 independent experiments. Gastroenterology 2011 140, 310-321.e4DOI: (10.1053/j.gastro.2010.10.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 (A) Expression of genes for adhesion molecules related to epithelial transmigration in pancreatic multistep carcinogenesis, analyzed by real-time reverse transcribed polymerase chain reaction. Significant value of *P < .05;**P < .01; and ***P < .001. (B) Immunohistochemical detection of ICAM2 protein in normal pancreatic duct (left photo), IPMA (center photo), and IPMC (right photo). Endothelial cells expressed ICAM2 (arrowhead). Expression of the ICAM2 was compared during pancreatic carcinogenesis (right). Ten cases per group were tested. Bars represent mean ± standard deviation. Differences between the 2 groups were examined for statistical significance using Student's t test. Significant value of *P < .0001. (C) Static binding assays detected that specific adhesion of CD14+ monocytes and monocyte-derived DCs to immobilized ICAM2. (Left and middle) Squares and diamonds represent no antibody-treated cells and anti-CD18 antibody-treated cells, respectively. Anti-CD18 antibody treatment inhibited the binding of monocytes to the immobilized ICAM2 almost completely. Data represent 1 of 5 independent experiments. (Right) Monocyte-derived DCs treated with or without CXCL17 were added to wells coated with human ICAM-2-Fc (12.5 μg/mL). Results show no antibody-treated cells (squares), anti-CD18 antibody-treated cells (diamonds), anti−DC-SIGN antibody-treated cells (circles), and cells treated with both anti-CD18 antibody and anti−DC-SIGN antibody (triangles). Either anti-CD18 antibody or anti−DC-SIGN antibody treatment inhibited the binding of monocytes to the immobilized ICAM2, and the binding of monocytes to ICAM2 was inhibited almost completely by treatment with both antibodies. Data represent 1 of 5 independent experiments. Differences between CXCL17-untreated and treated groups were examined for statistical significance using Student's t test. *P < .05 is significant. Gastroenterology 2011 140, 310-321.e4DOI: (10.1053/j.gastro.2010.10.009) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 (A) Chemokine (C-X-C motif) ligand 17 (CXCL17) and/or intercellular adhesion molecule 2 (ICAM2) expression in CMS5a tumor cells reduced tumor development. Balb/c mice were injected with parent (black circles), CXCL17-expressing (red or pink crosses), ICAM2-expressing (blue or light blue triangles), or both CXCL17 and ICAM2-expressing (green or dark green clear squares) CMS5a cells. Each data point represents the mean tumor size ± standard deviation of 5 mice. Significant value of *P < .01 and **P < .001 (compared to parent tumor cells). (B) Tumor-infiltrating CD3+, CD4+, and CD8+ T cells and CD11b+CD11c+ dendritic cells (DCs) in parent (white columns), CXCL17-expressing (red columns), and ICAM2-expressing (blue columns) CMS5a tumors. Columns and bars show mean (n = 6) and standard deviation, respectively. Significant value of *P < .05 and **P < .01 (compared to parent tumor cells). (C) Retardation of CXCL17- and/or ICAM2-expressing tumor growth was mediated by cellular immune response. Balb/c mice were injected with parent (top), CXCL17-expressing (second), ICAM2-expressing (third), or both CXCL17 and ICAM2-expressing (bottom) CMS5a cells. We used CD4+ T cell-depleted (red), CD8+ T cell-depleted (blue), and control antibody-injected (black) mice as recipients. Five mice per group were used. Significant value of *P < .05 and **P < .01 (compared to tumor growth in mice injected with control antibody). (D) Splenic lymphocytes from mice either bearing parent CMS5a tumors (open circle, closed triangle) or having rejected primarily CXCL17- and ICAM2-expressing CMS5a tumors and secondary parent CMS5a tumors (open square, closed diamond, open diamond) were cocultured with irradiated CMS5a cells for 5 days and then tested for cytotoxic T-cell assay against 51Cr-labeled ICAM2-expressing CMS5a cells (open square, open circle), parent CMS5a cells (closed diamond, closed triangle), or CT26 cells (open diamond). Each point represents the mean of triplicate cultures and the bars represent standard deviation. Differences of results in the experiments using lymphocytes from between mice bearing tumors and mice having rejected tumors were examined for statistical significance using Student's t test. Significant value of P < .01 (* or **). (E) Splenic lymphocytes from mice having rejected primarily both CXCL17- and ICAM2-expressing CMS5a tumors and secondary parent CMS5a tumors were cocultured with irradiated CMS5a cells for 5 days and then tested for cytotoxic T-cell assay against 51Cr-labeled ICAM2-expressing CMS5a cells with anti-ICAM2 antibody (closed diamond) or isotype-matched antibody (open square). Significant value of *P < .01. Gastroenterology 2011 140, 310-321.e4DOI: (10.1053/j.gastro.2010.10.009) Copyright © 2011 AGA Institute Terms and Conditions