Fig 2: Pharmacologically inhibiting mTORC1 activity leads to the increased expression of a FAK signature gene set. (A) Analysis of FDS expression in breast cancer subtypes using data from the TCGA Pan-Cancer Atlas. Groups with the same letter are not statistically different; groups with different letters have a p-value < 0.05. Red lines represent medians. Dashed lines within the violin plots represent quartiles. (B) Kaplan–Meier plots of the overall survival of breast cancer patients whose tumors had high (top 5%, n = 54) or low (bottom 95%, n = 1028) expression of the FDS. Data were obtained from the TCGA Pan-Cancer Atlas. Log-rank test, p < 0.05. (C) GSEA plot and (D) heatmap of genes in FDS evaluated in RNA-seq data from MDA-MB-231 orthotopic xenografts treated with vehicle or rapamycin for three days.

Fig 3: Targeted ablation reveals unique roles for PSC-derived CAFs in regulation of the ECM and mechanosignaling. A, IHC staining and quantification of GFP+ cells in normal pancreas tissue from Fabp4-Cre;Rosa26mTmG mice and from Rosa26mTmG/iDTR mice 7 days after intraductal injection with AAVKP1-Fabp4-Cre (n = 5). Data are presented as mean ± SEM. Scale bar, 50 µm. B, Schematic of tumor modeling using intraductal injection of AAVKP1-Fabp4-Cre and orthotopic transplantation of KPC PDAC cells into Rosa26mTmG/iDTR hosts. C, IHC staining for GFP, PDPN, and a-SMA of KPC FC1199 PDAC in Rosa26mTmG/iDTR hosts with intraductal injection of AAVKP1-Fabp4-Cre, enrolled when tumors reached 5 to 6 mm in diameter and treated with PBS or DT for 5 days (n = 4). Scale bar, 20 µm. D, IHC staining for TNC of KPC FC1199 PDAC in AAVKP1-Fabp4-Cre–injected Rosa26mTmG/iDTR hosts, enrolled at 5 to 6 mm in tumor diameter and treated with PBS or DT for 5 days (n = 3). Scale bar, 50 µm. Data are presented as mean ± SEM. E, IHC staining for p-MLC2 of PDAC samples as described in D. Scale bar, 50 µm. Data are presented as mean ± SEM. Quantification (F) and images (G) of fibrillar collagen content analyzed by second harmonic generation with normalized intensity as a quantification of concentration in control and PSC-depleted PDAC (n = 3 per group). Scale bar, 42.5 µm. H, Force maps generated by atomic force microscopy (AFM) on KPC FC1199 PDAC in AAVKP1-Fabp4-Cre–injected Rosa26mTmG/iDTR hosts (n = 3 per treatment group, control: 1,063 data points, depleted: 717 data points), excised after 5 days of treatment with PBS or DT. I, Quantification of Young's modulus per AFM measurements on control and PSC-depleted PDAC as described in F. The dashed line on the graph to the right denotes the approximate stiffness of normal murine pancreas tissue. J, IHC staining for p-FAK (Y397) of control and PSC-depleted PDAC harvested after 5 days of depletion (n = 3). Scale bar, 50 µm. Data are presented as mean ± SEM. K, Kaplan–Meier plot depicting overall survival of patients with PDAC with high versus low expression of a PSC-derived CAF ECM gene signature comprising 99 genes (see Methods), plotting the upper versus lower quartile (n = 73 per arm). *, P < 0.05; ****, P < 0.0001 by unpaired t test; ns, not significant.

Fig 4: LPA induces formation of actin stress fibers and phosphorylation of MLC and FAK in HTM cells. Serum-starved HTM cells (24 hours) treated with LPA (20 µM for 2 and 6 hours) showed significant increase in the levels of phosphorylated MLC (p-MLC; panel [A]) and FAK (p-FAK; panel [C]) compared with the control cells, based on quantitative immunoblot analyses. Values are mean ± SEM. n = 4, *P = 0.05. Changes in p-MLC and p-FAK were normalized to total MLC and GAPDH, respectively. These LPA-induced changes in cell contractility and adhesion were also associated with increased actin stress fiber formation, as evaluated by phalloidin fluorescence staining (B). Scale bar: 20 µm.

Fig 5: Cell culture in RAD16-I matrix functionalized with collagen type I. (a) SDS-PAGE of RAD or RAD/COL cell-free gels; (b) phase-contrast microscopy images of fibroblast 24 h after cell embedding within 0.15% RAD or RAD/COL gels. Scale bars represent 50 µm; (c) Western blot bands of FAK and Erk1/2 and its phosphorylated forms in PANC-1 cells cultured in 0.15% (soft) and 0.5% (stiff) RAD16-I gels mixed with increasing amounts of collagen I; (d) densitometry of pFAKY397/FAK shown in (c). GAPDH was used as loading control. Statistical differences are indicated as ** for p value < 0.01, *** for p value < 0.001 and **** for p value < 0.0001.