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, vivo experiments was performed using the STATA 11.0 software (StataCorp., College Station, TX) as previously described

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, RT-qPCR analysis of NRG1 mRNA levels in pancreatic tumor samples from patients who underwent surgical resection. Results are expressed as mRNA Arbitrary Units and normalized to the expression of the housekeeping gene HPRT. (B) NRG1?1 expression analysis by RT-qPCR and ELISA in the pancreatic cancer cells lines of AsPC-1, BxPC-3, CFPAC, HPAC, Mia-PaCa-2 and SW1990 and in AsPC-1-NRG1 cells that stably overexpress NRG1 and their conditioned medium

, AsPC-1-NRG1 and AsPC-1-Mock cells were xenografted in nude mice. Tumor growth was monitored every week, and at 40 days post-graft mice were sacrificed and tumors weighted (bottom panel). (D) ?SMA and vimentin expression by immunofluorescence analysis in CAFs from pancreatic cancer surgical biopsies or normal pancreatic fibroblasts (NPF) (top panels). (E) NRG1 mRNA was quantified by RT-qPCR in CAF cultures. Results are expressed as mRNA Arbitrary Units and normalized to HPRT expression. (F) The level of NRG1 secreted was, NPC are a mix of three normal pancreatic cells. NPF are normal pancreatic fibroblasts

, These CM was also added to serum-starved BxPC-3 cells for 15 minutes. The total level of phosphorylated HER3 was then measured by Western blotting

*. , , vol.001

, Figure 2. Characterization of the Ig-like domain-specific antibody 7E3

, HER3-Fc was captured on the sensor chip surface using an antihuman Fc antibody, then 185nM rhNRG1?1-ECD was injected followed by an injection of 200nM of 7E3 antibody. (B) Immunofluorescence detection of the 7E3 antibody on HER3-positive (shLuc) or HER3-silenced shHER3 BxPC-3 cells. Cells were serum-starved and then incubated with increasing concentrations of rhNRG1?1-ECD (1 to 100ng/ml) and 20 ?g/ml of 7E3 at 4°C for 1hour. Then, FITCconjugated goat anti-mouse IgGs were added. Anti-HER3 and anti-CEA antibodies were used as controls for HER3 and CEA expression. (C) Alanine scanning analysis to precisely identify 7E3 binding motif in the three membrane pentadecapeptides that correspond to the identified antibodyimmunoreactive sequences. Each bar represents 7E3 reactivity toward a pentadecapeptide sequence in which the indicated amino acid was substituted by alanine. The mean spot reactivity for, SPR analysis of the binding kinetics between the anti-NRG1 antibody 7E3, rhNRG1?1-ECD and recombinant human HER3-Fc

, Cells were first incubated or not with 50ng/ml rhNRG1?1-ECD and/or with 50µg/ml 7E3 or with the anti-HER2 antibody trastuzumab (positive control) at 37°C for 30min. Then, cells were incubated with hPBMC (37°C) at an effector:target ratio of 15:1 for 24h. BxPC-3 cell lysis was evaluated by measuring LDH release by bioluminescence

, Figure 3. The 7E3 antibody inhibits NRG1?1-induced phosphorylation of HER3 and downstream signaling pathways and promotes HER3 downregulation

, After cell lysis, the expression of phosphorylated and total HER3, AKT and ERK was analyzed by western blotting. (B) 7E3 effect on HER4 phosphorylation was studied by western blotting in IGROV-1 cells following the same protocol as in A. (C) Internalization of 7E3 in BxPC-3 cells was detected by immunofluorescence

, 4°C or 37°C) for different time points and then with FITC-conjugated goat anti-mouse IgGs, or with 7E3 directly labelled with FITC, Control cells were incubated with FITC-conjugated goat anti-mouse

. Iggs, D) HER3 expression in BxPC-3 cells was analyzed by Western blotting at different time points after incubation with 100 µg/ml 7E3 or irrelevant antibody (IR) together with 15ng/ml rhNRG1?1-EDC and was quantified relatively to tubulin levels

, Figure 4. The 7E3 antibody inhibits viability, growth and migration of pancreatic cancer cells

, After 10 days of growth in the presence of 50ng/ml rhNRG1?1-EDC and 50-100µg/ml 7E3 or irrelevant antibody (IR), spheroid cells were labeled with (A) BxPC-3 cells and CAFs (both NRG1-positive) were injected at a 1:2 ratio in the pancreas of athymic mice (n=10 per group), Cell viability of BxPC-3 and HPAC cells was analyzed by using the sulforhodamine B assay. After serum starvation for 24h, cells were incubated for 5 days with 15 or 10 ng/ml rhNRG1?1-EDC, respectively, and with different concentrations of 7E3 or irrelevant antibody (IR)

*. , 001). (C) Immunohistochemistry analysis shows the presence of ?SMA positive fibroblast and an increase of cleaved caspase3 in 7E3 treated tumors