Figure 2.

Putative OIS biomarker expression is unchanged in mPanIN, conditionally mutant for p53 or Cdkn2a. All animals were housed in accordance with the UK Home Office, within the designated vivarium at the Cancer Research UK Cancer Research Institute. Pancreatic-specific expression of a conditional point-mutated allele of Kras (LSL-Kras^G12D) elicits mPanIN and PDA that exhibit pathology, consistent with the human condition (Hingorani et al., 2003). Acceleration of tumor development was accomplished by interbreeding these Kras^G12D;Cre (KC) mice with mice harboring a conditional floxed allele of Trp53 or Ink4A/ARF to generate the Kras^G12D;p53^flox/flox;Cre (KPPC) or Kras^G12D;Ink4A/ARF^flox/flox;Cre (KIIC) strains, respectively. Rosa26-RYFP allele (Srinivas et al., 2001) was bred to KC mice to label cre-mediated recombination in the pancreas. Archival formalin-fixed paraffin-embedded KC, KPPC and KIIC pancreata were sectioned for immunohistochemical analyses. (a) Proliferation of KC, KPPC and KIIC PanIN samples. Each data point represents the average of at least five fields from a single mouse. Statistical analyses were carried out using Graphpad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Mann–Whitney U-test was used to determine significance: KC versus KPPC, P = 0.0400; KC versus KIIC, P = 0.0029. (b) KPPC and KIIC mice harbor more high-grade PanIN, compared with KC mice. (c) Immunohistochemical analysis of potential OIS biomarker expression in mouse. Knockout mouse tissue was used as a negative control to evaluate the specificity of all markers, except DcR2. DcR2 negative control is species-specific IgG (top row). Arrowheads denote positive cells. Scale = 200 μm. (d) Quantification of potential OIS biomarker expression in murine KC PanIN, and KPPC and KIIC PDA. At least three representative images from each mouse were quantified, the average of which represents a single data point. (e) Immunofluorescent detection reveals that Dec1, DcR2, γH2AX and p19^ARF (green) can be colocalized in Ki67 (red)-positive epithelial cells in mPanIN and PDA. Scale = 10 μm. White arrows denote double positive cells. (f) Quantification of coimmunofluorescent staining indicates that a significant fraction of Ki67-positive cells harbor putative OIS biomarkers. Error bars indicate s.e.m. (N = at least 4). At least three representative images from each mouse were quantified, the average of which represents a single data point.