Volume 139, Issue 1, Pages 292-303 (July 2010) Dasatinib Inhibits the Development of Metastases in a Mouse Model of Pancreatic Ductal Adenocarcinoma  Jennifer P. Morton, Saadia A. Karim, Kathryn Graham, Paul Timpson, Nigel Jamieson, Dimitris Athineos, Brendan Doyle, Colin McKay, Man–Yeung Heung, Karin A. Oien, Margaret C. Frame, T.R. Jeffry Evans, Owen J. Sansom, Valerie G. Brunton  Gastroenterology  Volume 139, Issue 1, Pages 292-303 (July 2010) DOI: 10.1053/j.gastro.2010.03.034 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Src expression and activity in human ductal adenocarcinoma. Immunohistochemical analysis of Src (left panels) and phospho-Src Y418 (right panels) in a tumor array of human PDAC. Representative images are shown for absence of staining, −; weak staining, +; moderate staining, ++; and strong staining, +++. Original magnification, 10×. Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 High Src expression and activity in human PDAC correlates with reduced survival. (A) Box plot of Src histoscore vs tumor grade. (B) Box plot of Src histoscore vs LNR. (C) Box plot of Src histoscore vs vascular invasion. (D) Kaplan–Meier analyses for high-grade patients (n = 34) showing that high Src expression is associated with poorer outcome. (E) Kaplan–Meier analyses for resection margin–positive patients (n = 90) showing that cases with evidence of grade 3 phospho-Src expression have poorer outcome after pancreaticoduodenectomy compared with those with grade 1/2 phospho-Src expression. Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Src expression and activity during development of PDAC in Pdx1-Cre, Z/EGFP, LSL-KrasG12D/+, LSL-Trp53R172H/+ mice. Immunohistochemical analysis of (A) Src and (B) phospho-Src Y418 in normal pancreatic ducts, PanINs, and PDAC. Images taken at magnifications of 40× for normal ducts and 20× for PanINs and PDACs. Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Dasatinib inhibits Src activity in mouse PDAC cells. (A) Cell lines were established from 3 PDACs (82739, 83320, and 86119) harvested from Pdx1-Cre, Z/EGFP, LSL-KrasG12D/+, LSL-Trp53R172H/+ mice. Phase contrast images are shown for each (left panels). Expression of Pdx1 in the cell lines confirms their pancreatic origin (right panels). Scale bars, 0.2 μm. (B) Recombination of Trp53R172H in cell lines. (C) PDAC cells were treated with a range of dasatinib concentrations for 24 hours before Western blot analysis using anti–phospho-Src Y418 and anti-Src antibodies. (D) PDAC cells were treated with a range of dasatinib concentrations for 96 hours and an MTT proliferation assay then was performed. Values are mean ± standard deviation of quadruplicate wells taken from a representative experiment in a series of 3. Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Dasatinib inhibits PDAC cell migration and invasion. (A) Migration of PDAC cells was determined in the absence or presence of dasatinib (100 or 200 nmol/L) using a wound healing assay. Values represent the wound area at the end of the experiment expressed as a percentage of the original wound area. Values are mean ± standard deviation of 3 areas of the wound and are taken from a representative experiment in a series of 3. (B) Western blot analysis of Src expression in PDAC cells treated with scrambled or Src siRNA sequences. β-tubulin is used as a loading control. (C) Migration of PDAC cells treated with scrambled or Src siRNA sequences calculated as in panel A. (D) Invasion of PDAC cells into collagen gels was determined in the absence or presence of 100 nmol/L dasatinib. Quantification of fold invasion greater than 20 μm relative to control (untreated cells) is presented from at least 3 independent experiments. Values are mean ± standard error. (E) PDAC cells were treated with a range of dasatinib concentrations for 24 hours before Western blot analysis using anti–phospho-FAK Y861 and anti–phospho-p130Cas Y249 antibodies. β-tubulin is used as a loading control. (F) MMP gelatin zymography was performed on supernatants of untreated and dasatinib-treated (100 nmol/L) cells. Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Determination of biologically active dose of dasatinib in mice. PDAC cells were grown as subcutaneous tumors, and the mice were treated with dasatinib at 5 mg/kg or 10 mg/kg. After 2 hours the mice were killed and tumors were collected. (A) Western blot analysis using anti–phospho-Src Y418 and anti-Src antibodies. (B) Immunohistochemical analysis of tumors stained with anti–phospho-Src Y418 antibody from vehicle and dasatinib-treated (10 mg/kg) animals. Images taken at 20× magnification. Representative results are shown for the 83320 cell line. Similar results were obtained for the 86119 and 82739 cells. Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Dasatinib does not increase survival but reduces the incidence of metastasis in Pdx1-Cre, Z/EGFP, LSL-KrasG12D/+, LSL-Trp53R172H/+ mice. (A) Kaplan–Meier survival curves of mice treated with vehicle or dasatinib (10 mg/kg) from 6 or 10 weeks of age. (B) The number of mice with metastases was counted at the time of death, and confirmed by histology, and results are presented as the percentage of the total number of mice in each cohort. Black columns represent mice with metastases and grey columns represent those without metastases. χ2 test: 6-week cohort vs vehicle, P = .048 and 10-week cohort vs vehicle, P = .036. (C) Lysates were prepared from tumors harvested from vehicle or dasatinib-treated (10 mg/kg) mice, and Western blot analysis was performed with anti–phospho-Src Y418 and anti-Src antibodies. (D) Immunohistochemical analysis of tumors harvested from vehicle and dasatinib-treated animals, stained with anti–phospho-Src Y418 antibody. Images taken at 20× magnification. In each case representative examples are shown from 2 individual tumors. (E and F) Cell proliferation was assessed by labeling cells for 2 hours with bromodeoxyuridine (brdu) before killing the mice. Immunohistochemical analysis of BrdU was performed, and the number of positive nuclei was determined. (E) Ten PanINs were scored from 4 mice in each treatment group (n = 40; Mann–Whitney U test, P = .665). (F) PDAC. Ten high-powered frames were scored from 4 mice in each treatment group (n = 40; Mann–Whitney U test, P = .557). Gastroenterology 2010 139, 292-303DOI: (10.1053/j.gastro.2010.03.034) Copyright © 2010 AGA Institute Terms and Conditions