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, B Twenty‐one and 38 days after syngeneic and orthotopic co‐grafting of mouse FAK‐WT or FAK‐KD fibroblasts plus tumour cells, mice were euthanized and tumour dissociated. Relative frequencies of tumour‐infiltrating immune cells (CD3, CD4, CD8, LB, NK, NKT and Treg) using flow cytometry at 21 days (A) and 38 days (B). All stained cells were analysed with a Fortessa X20 flow cytometer (BD Biosciences), and data processed using FlowJo software (Beckman Dickinson). Values are means ± SEM from 5 to 10 mice per group, unpaired two‐tailed Student's t ‐test. C, D Quantification of CD206 (C)‐ and CD8 (D)‐positive cell frequency (based on IHC staining) on whole pancreas, tumoural, adjacent and fibrotic areas with representative immunochemistry pictures of each staining on different areas. Values are means ± SEM from 9 to 10 mice per group. * P < 0.05 using unpaired two‐tailed Student's t ‐test. Scale bar: 50 μm E Mouse embryonic fibroblasts were incubated with conditioned medium (CM) from pancreatic cancer cells during 24, 48 or 72 h before being lysed (CM were harvested from tumour cells 72 h after tumour cells were seeded in F12 medium plus 0.5% FBS). PDGFR‐α, FAP‐α, α‐SMA and GAPDH expressions were evaluated by Western blot. F Validation of the BMDM‐derived macrophage polarization into M1 or M2. Left: schematic of the experiment; right: percentage of M1‐ or M2‐positive cells. Results were acquired by flow cytometry and processed using FlowJo software. *** P < 0.001, by Bonferroni's multiple comparison test; values are mean ± SEM from three experiments. G Evaluation of the impact of FAK inhibitor that has been pre‐incubated for 48 h at 37°C in DMEM/F12 + 0.5% FBS on macrophage migration. M, medium DMEM/F12 + 0.5% FBS; CM, conditioned medium. Quantification performed using Cell Observer videomicroscope (Zeiss) and analysed with ImageJ. * P < 0.05, by Tukey's multiple comparison test; values are mean ± SEM from three experiments.

Article Snippet: Small tissue blocks were cut (0.5–1 mm 3 ) using razor blade and seeded in 10‐cm 2 uncoated culture wells in the presence of Dulbecco's modified Eagle's medium F12 (DMEM/F12 Lonza D8437) containing 10% FBS (Life Technologies) supplemented with l ‐Glutamine ( l ‐glu, 2 mmol/l), penicillin and streptomycin (P/S).

Techniques: Flow Cytometry, Staining, Software, Two Tailed Test, Immunohistochemistry, Incubation, Western Blot, Biomarker Discovery, Derivative Assay, Comparison, Migration