Volume 138, Issue 4, Pages 1595-1606.e8 (April 2010) Netrin-1 Mediates Early Events in Pancreatic Adenocarcinoma Progression, Acting on Tumor and Endothelial Cells  Laurent Dumartin, Cathy Quemener, Hanane Laklai, John Herbert, Roy Bicknell, Corinne Bousquet, Stéphane Pyronnet, Vincent Castronovo, Martin K. Schilling, Andreas Bikfalvi, Martin Hagedorn  Gastroenterology  Volume 138, Issue 4, Pages 1595-1606.e8 (April 2010) DOI: 10.1053/j.gastro.2009.12.061 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Overview of the PDAC–CAM model. (A) Human pancreatic tumor cells BxPC3 are grafted on the intact CAM surface at E10 (T0) of embryonic development. On the second day (T2) after implantation, tumor cells start their invasion across the epithelial sheet. From T4, tumor nodes densely encircled by CAM vessels are established in the CAM stroma. Tumor development and stromal response can be followed at the molecular and morphologic levels during the 8 days after cell grafting. (B) Biomicroscopy images of the successive tumor progression steps. (C and D) Kinetic follow-up of tumor progression by measure of total invasion area (C) or dosage of the human CA19-9 tumor marker in chick embryo serum (D). Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Morphologic characteristics of the PDAC–CAM model. (A–C) Top views of pancreatic tumor cell clusters (*) progressively and collectively invading through the epithelial surface of the CAM. (D) Cryosection of the CAM (dashed line, CAM surface) showing tumor cells (*) collectively invading the stroma underneath the epithelium (arrowed line shows capillaries from the CAM plexus around the invasive tumor cluster). (E and F) Vascularized tumor nodes in the CAM-stroma (circle, individually migrating tumor cell). (A–C) Scanning electron microscopy. (D–F) Epifluorescence microscopy. Scale bars, 100 μm. T, tumor day; CK19, cytokeratin 19; SNA, sambucus nigra lectin; CTB, CellTracker Blue. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Simultaneous transcriptomic analysis of tumor and CAM stroma gene expression. (A) Gene expression was compared between early (T1) and late (T6) stages of tumor development with human (tumor genes) and chicken (CAM stroma genes) Affymetrix GeneChips. Stroma genes were submitted to a computational ortholog screen aiming at identifying putative human ortholog genes. (B) Distribution of >2-fold regulated genes in microarray analysis. Chicken genes are divided into genes with or without putative human ortholog. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Netrin-1 acts both on tumor and ECs in vitro. (A) Effect of netrin-1 protein on tumor and EC apoptosis. Apoptosis was induced by gemcitabine treatment (BxPC3 cells) or serum starvation (HUVECs), and the effect of increasing concentration of netrin-1 protein (or VEGF) on apoptosis was determined. CM, culture medium. (B) Adhesion assay on ECM proteins or netrin-1–coated surfaces. Adhesion level on bovine serum albumin–coated wells represents experimental background. (C) Boyden chamber invasion assay on BxPC3 cells. Tumor cells were incubated 30 minutes in serum-free medium with or without netrin-1 before seeding on Matrigel matrix. Tumor cells were allowed to invade during 24 hours with serum-free medium supplemented with 1% fetal bovine serum as a chemoattractant in the lower chamber. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Silencing of NTN1 expression in tumor cells inhibits invasion in the PDAC–CAM model. (A) Anti–netrin-1 immunolabeling of a T6 tumor node. (B) Levels of NTN1 and CEACAM6 mRNA in BxPC3 cells 24 hours after transfection with specific siRNA. (C) Kinetic follow-up of total invasion area of BxPC3 cells transfected with different siRNAs. (D) Representative images of invasive tumor cell clusters in T2 and T6 CAM grafted with siRNA-transfected BxPC3 (magnification ×30). CO, control. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Netrin-1 is overexpressed at the mRNA and protein level in human PDAC samples. (A) Relative expression of NTN1 in pancreatic adenocarcinoma biopsies of 30 human patients analyzed by semiquantitative real-time RT-PCR. Three groups of overexpression can be distinguished. (B and C) Immunohistological analysis of netrin-1 expression in normal pancreas and PDAC. (D) Western blot analysis confirmed expression of netrin protein in 9/10 PDAC samples. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Modification of pericyte coverage on blood vessels. (A) Top view of CAM vasculature surrounding a T2 tumor node. Arrows delimitate frontier between 2 zones of different pericytes abundance. (B and C) Internal view within T2 and T6 nodes, respectively. Arrows indicate pericytes on blood vessels. Scale bars =100 μm. T, tumor day; CK19, cytokeratin 19; SNA, sambucus nigra lectin; CTB, CellTracker Blue. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Real-time PCR confirmation of microarray analysis. Expression of several up-regulated tumor and stroma genes, according to microarray data, were analyzed by semiquantitative real-time PCR compared with human S16 gene by using specific primers for each gene. S16 gene was used as a reference gene because it shows a weak variability at the mRNA level between early and late stages of tumor invasion. Dynein gene, showing no variability of expression according to microarray data, was also used as a reference gene with similar results (data not shown). (A) Analysis of tumor and stroma gene expression between T1 and T6. (B) Comparison of selected stroma gene expression at day 16 of embryonic development between normal CAM (E16) and implanted CAM (T6). Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Expression of netrin-1 and its receptors in human pancreatic cancer cells and ECs. (Upper table) Gene expressions were analyzed by real-time PCR compared with housekeeping gene S16. Levels of expression were determined by the ΔCt method (Ctgene of interest – CtS16). No expression (−); weak expression (+/−); moderate expression (+); strong expression (++); high expression (+++). (Lower panel) Western blot analysis of netrin-1 protein and the UNC5B receptor in indicated cell lines. HPDE, human pancreatic ductal epithelial cells. Gastroenterology 2010 138, 1595-1606.e8DOI: (10.1053/j.gastro.2009.12.061) Copyright © 2010 AGA Institute Terms and Conditions