Fig 1: In vitro model and axon guidance molecule expression in vitro. (a) Graphical representation of the technical procedure used to mimic in vitro the PDA intratumoral compartment and to produce conditioned media (Cd media). (b) IF images of aSMA staining on human fibroblasts alone (F) or cocultured with macrophages (FcoM), human PDA (PDA) or CAF from human PDA (magnification: × 20 and × 40 for in vivo and in vitro images, respectively). (c) Representative images of fibroblasts (F) or fibroblasts cocultured with macrophages (FcoM) labeled for SLIT2 (green). SLIT2 quantification from IF staining on fibroblasts (F, used as normalizer) or fibroblasts cocultured with macrophages (FcoM) (n=3; **P<0.01). (d) Human mRNA level of some genes composing the ‘axon guidance' family was analyzed by quantitative real-time PCR (QRT-PCR) in human pancreatic tumoral cell lines (Miapaca2 used as normalizer, except for ROBO1, which are not expressed in Miapaca2, and Panc1), human fibroblasts (F) and human fibroblasts cocultured with mouse macrophages (FcoM). Light green, P<0.05 versus Panc1; dark green, P<0.05 versus F (n=3). Md, conditioned media from: SNF, sNF 96.2; F, fibroblasts; M, macrophages; F+M, mixed Md from separated cultures of fibroblasts and macrophages; FcoM, cocultures of fibroblasts and macrophages
Fig 2: SLIT2 modulates N-cadherin (N-Cadh)/ß-catenin (ß-cat) signaling to influence Schwann cell migration ability. (a) N-cadherin/ ß-catenin binding was analyzed by co-immunoprecipitation (IP) in SNF cells after incubation with F+M (control) or FcoM media. (n=3). (b) N-cadherin/ß-catenin binding was analyzed by co-immunoprecipitation in SNF cells after incubation with F+M (control) or FcoM media from fibroblasts transfected with Ctr (Si-Ctr) or SLIT2 (Si-SLIT2) siRNA. (n=3). (c) Nuclear extracts from SNF cells incubated with SNF, F+M or FcoM media (with fibroblasts transfected with Si-Ctr or Si-SLIT2 siRNA) were analyzed for ß-catenin expression. Lamin A/C was used as a loading control and ß-tubulin was used asa quality control of nuclear extracts. ß-Catenin expression was normalized to respective lamin A/C expression (n=3). (d) mRNA level of three ß-catenin targets (C-MYC, LCF4 and MMP9) analyzed by quantiative real-time PCR (QRT-PCR) in SNF cells after incubation with SNF (used as normalizer), F+M or FcoM media (n=3; *P<0.05; **P<0.01; ***P<0.001). Md, conditioned media from: SNF, sNF 96.2; F, fibroblasts; M, macrophages; F+M, mixed Md from separated cultures of fibroblasts and macrophages; FcoM, cocultures of fibroblasts and macrophages
Fig 3: SLIT2 expression pattern in CAFs of the stromal compartment within human (h) and mouse PDA. (a) IHC and IF images of human PDA stained for SLIT2 (upper left), SLIT2 (red) and cytokeratin 19 (CK19) (green) in the upper right and SLIT2 (red) and aSMA (green) in the lower left. Fluorescence intensity quantification (of IF staining) was carried out on four human PDA (magnification: × 20) (**P<0.01) using Image J software. (b) SLIT2 mRNA quantification by quantitative real-time PCR (QRT-PCR) in CAFs extracted from seven different human PDA versus human pancreatic tumoral cell lines, Miapaca2 (Mia) as reference (value at 0.00001e5), Panc1 (Pc1) and Capan-2 (Cp2) (***P<0.001). (b, inset) IF representative images of CAFs stained for SLIT2 (green) and aSMA (red) (magnification: × 20). (c) Western blot of SLIT2 expression in protein extracts from human PDA or healthy human pancreas (demonstrating the increased expression of SLIT2 in PDA extracts). Actin was used as a loading control. (d) SLIT2 mRNA quantification by QRT-PCR in the pancreas of 8-week-old healthy mice (KrasG12D/Ink4Af/f) and PDA-bearing mice (pdx1-cre/KrasG12D/Ink4Af/f) (**P<0.01). (e) SLIT2 expression and HE counterstaining of pancreas from 8-week-old healthy mice and PDA-bearing mice (upper images) and SLIT2 staining (lower images). *, Epithelial/ductal tumoral cells/structures; arrows, stromal/fibroblast compartment. (f) Representative IF images of CAFs labeled with aSMA (red) and SLIT2 (green) in mouse PDA (magnification: × 20; scale bar: 100 µm)
Fig 4: SLIT2 is positively correlated with PANR in vivo. (a) Correlation between aSMA expression and SLIT2 level in pancreatic tumor samples from eight PDA-bearing mice. (b) Correlation between Slit2 score and the number of intratumoral nerves (It-tum nerve) in pancreatic tumor samples from 12 PDA-bearing mice. (c) Correlation between SLIT2 level and number of intratumoral nerves (It-tum nerve) in 15 human PDA xenograft samples. (d) Count of nerves positive for Ki67 staining in Schwann cells, using 10 mouse PDA samples with high (n=5) or low (n=5) SLIT2 level
Fig 5: SLIT2/ROBO pathway impacts on neural cells behaviors linked to PANR. (a–d) Migration assay: human Schwann cells were assessed for migration abilities on Boyden chamber assay for 4 h with (a) various conditioned media±SLIT2 antibody (Slit2-Ab) to deplete SLIT2 from the conditioned media±blocking peptide (SLIT2-BP) as control. (b) Conditioned media from control (SNF), mixed (F+M) or cocultures (FcoM) using fibroblasts transfected with control or SLIT2 targeting siRNA. (c) SNF conditioned medium supplemented, or not, with 25 pg of human recombinant SLIT2. (d) Conditioned medium from control (SNF), mixed (F+M) or cocultures (FcoM) applied on Schwann cells transfected with control or Robo1 (A and B) or Robo2 (A and B) targeting siRNA. (a–d) (n=3; *P<0.05; **P<0.01; ***P<0.001). Md, conditioned medium from: SNF, sNF 96.2; F, fibroblasts; M, macrophages; F+M, mixed Md from separated cultures of fibroblasts and macrophages; FcoM, cocultures of fibroblasts and macrophages
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