Journal: EMBO Molecular Medicine

Article Title: FAK activity in cancer‐associated fibroblasts is a prognostic marker and a druggable key metastatic player in pancreatic cancer

doi: 10.15252/emmm.202012010

Figure Lengend Snippet: A Representative images of CK19 and α‐SMA immunohistochemistry and Sirius red staining in serial sections of primary mouse tumour (graft of FAK‐WT or FAK‐KD fibroblasts + tumours cells at 38 days after injection) at the invasive front. Scale bar, 100 μm. Bottom: schematic organization of the cells (tumour cells in brown, CAFs in yellow and non‐transformed epithelial cells in beige). B Representative images of ECM deposition (stained using Alexa Fluor 488 succinimidyl ester [NHS]) generated by FAK‐WT or FAK‐KD activated fibroblasts during migration. Scale bar, 50 μm. C Quantification of collagen I gel contraction (calculated based on photographed gels using the formula 100*(well diameter − gel diameter)/well diameter) induced by fibroblasts in presence or not of tumour cells (left: one‐way ANOVA with Tukey's method for multiple comparison), or by hCAFs in presence or not of FAK inhibitor (FAK‐I, 1 μM; right: paired two‐tailed Student's t ‐test for each time). Values are means ± SEM from three independent experiments of triplicates. * P < 0.05, ** P < 0.01, *** P < 0.001. D Quantification of the percentage change of Y397 FAK, total FAK, collagen I (coll I), collagen III (coll III), collagen IV (coll IV), LOXL2, periostin (POSTN), osteopontin (OPN) expression by human CAFs treated or not (NT) with FAK inhibitor (1 μM), based on immunofluorescence analyses. Values are means ± SEM obtained from at least five primary hCAFs, isolated from 5 to 12 different fresh human PDAC samples. NS: not significantly different; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 using paired two‐tailed Student's t ‐test. E Collagens I, III and IV and periostin deposition are shown using heat map of the IF staining, and cell shapes are identified by white line. Scale bar, 50 μm. F–K Characterization of the ECM‐enriched fraction (matrisome analyses) from hCAFs treated or not with FAK‐I by LC‐MS/MS proteomics. (F) Pie charts display the relative proportion of each of the major classes of matrisome proteins in non‐treated hCAFs ( N = 3 biological replicates with two technical replicates/condition) vs FAK-I‐treated hCAFs ( N = 3 biological replicates with two technical replicates/condition). (G, H) Left: Normalized peptide abundance for collagens (G) or proteoglycans (H) from hCAFs (each CAF duplicate is represented, CAF1 in blue, two in red and three in black) treated or not with FAK inhibitor. Right: Heatmap of the collagen (G) or proteoglycan (H) modifications in the three hCAFs upon FAK inhibition in hCAFs. The value of log 2 fold change between FAK inhibitor‐treated CAFs and non‐treated CAFs for each protein was indicated by the coloured scale, with red indicating increased expression while blue implies decreased expression. For each CAFs and each protein, log 2 fold change was calculated based on two technical replicates per condition (biological replicate n = 3, technical replicate n = 2). (I) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK‐I. (J) Heatmap of the glycoprotein modifications upon FAK‐I in hCAFs. (K) Normalized peptide abundance for “matrisome‐associated” protein division: ECM‐affiliated proteins, secreted factors and ECM regulators from hCAFs treated or not with FAK inhibitor. (G, H, K) Each individual patient‐derived activated fibroblast was assigned to a specific symbol (see Table EV2 ). L Left: Representative pictures of β1 integrin activation (red) of tumour cells, upon adhesion to either ECM deposited from non‐treated CAFs or treated with FAK‐I for 7 days, analysed by IF. Merge: activated β1 integrin (red), phalloidin (green), dapi (blue). Right: quantification of the number of activated integrin β1 cluster per cells and size of those clusters. Values are means ± SEM, **** P < 0.0001 using paired two‐tailed Student's t ‐test on at least nine images (of three to 33 cells per image) per group, scale bar: 10 μm. Source data are available online for this figure.

Article Snippet: CAFs were isolated from human pancreatic tumour tissues using the outgrowth method described by Bachem et al ( ).

Techniques: Immunohistochemistry, Staining, Injection, Transformation Assay, Generated, Migration, Comparison, Two Tailed Test, Expressing, Immunofluorescence, Isolation, Liquid Chromatography with Mass Spectroscopy, Inhibition, Derivative Assay, Activation Assay