Figure 8.

PDAC heterogeneity and transcriptional subtype impact FAK function. (A) Graphical summary showing that single-cell cloning can result in a panel of cell lines broadly representing the heterogeneity of the original parental population. (B) Representative anti-FAK western blots showing FAK re-expression in a panel of clonal cell lines in which CRISPR-Cas9 has been used to delete FAK expression. (C) NanoString nCounter gene expression data from IFNγ stimulated cells generated using a mouse PanCancer Immune Profiling panel. Data represented as fold change in gene expression relative to FAK-wt counterpart. (D) Heat map of FAK-/- GSVA scores for expression of genes associated with classical and squamous subtypes of pancreatic cancer. (E) Representative anti-GATA6, HNF1A, HNF4A, FOXA2 and PDX1 western blot from whole cell lysates isolated from six FAK-/- clonal cell lines. Anti-GAPDH used as a loading control. (F) Western blot showing CRISPR-Cas9 deletion of HNF1A, GATA6 and HNF1A+GATA6 in FAK-wt and FAK-/- cells. Anti-GAPDH used as a loading control. (G) Relative quantification of Psmb8 and H2-Kb gene expression using qRT-PCR following stimulation with IFNγ. n=3. (H) Relative quantification of Psmb8 and H2-Kb expression using qRT-PCR in FAK-wt and FAK-/- cells±treatment with TGFβ1 for 9 days. Cells were stimulated for 24 hours with IFNγ ± TGFβ1 prior to RNA extraction. n=3 (I) Relative quantification of Psmb8 and H2-Kb expression using qRT-PCR in FAK-wt and FAK-/- cells±treatment with TGFβ inhibitor for 2 weeks. Cells were stimulated for 24 hours with IFNγ ± TGFβ inhibitor prior to RNA extraction. n=3. IFNγ treatment was 10 ng/mL for 24 hours (C, G, H, I). Data in G–I represented as mean±SEM. Statistical significance calculated using a one-way ANOVA with Tukey’s multiple comparison test. *p≤0.05, **p≤0.01. ANOVA, analysis of variance; FAK, focal adhesion kinase; GSVA, gene set variation analysis; PDAC, pancreatic ductal adenocarcinoma.