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Fig 1.

ROCK1 protein expression and gene amplification in PDAC tissues.

A) Immunostaining of ROCK1 protein in a normal pancreas section (negative staining) and a PDAC tissue section (strong staining with a score of 3+). Black arrow: tumor cells; oval arrow: cancer associated fibroblasts. B) aCGH plots of the whole genome (bottom panel), chromosome 18 (middle panel) and the ROCK1 gene locus (top panel) from a representative patient with a focal (1.76Mb) 18q11.1 amplicon. Blue shaded areas denote aberrant copy number intervals defined by ADM2 step gram algorithm [31] with a log2ratio of +1.4 (p<0.001) for the ROCK1 containing amplicon.

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Table 1.

Summary of immunohistochemical staining of ROCK1 in PDAC tissue microarrays

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Fig 2.

Knockdown of ROCK1 by siRNA in pancreatic cancer cell inhibits cell proliferation.

A) ROCK1 was detected in pancreatic cancer cell lines (PANC-1, Mia PaCa-2, SU.86.86, BxPC3, AsPC-1, and HS766T), the immortalized normal pancreatic ductal epithelial cell line (HPDE6), and the cancer associated fibroblasts (CW-1) by Western blotting. B) Western blotting analysis of ROCK1 knockdown by siRNA over the course of 72 hours. (C) Western blotting analysis of ROCK1 knockdown by siRNA (72 hour treatment) in two cell lines, SU.86.86 and PANC-1. (D) Growth curves of pancreatic cancer cells (PANC-1 and SU.86.86) treated with siRNA to ROCK1. * P < 0.001 (compared to the untreated control). UT, untreated; tR, transfection reagent only; NT, non-targeting; ASD, cell death (positive) control; R1, ROCK1 siRNA1; R2, ROCK1 siRNA 2.

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Fig 3.

Effects of the ROCK1 inhibition on pancreatic cancer cells and cancer-associated fibroblasts.

A) Fasudil dose response curves in pancreatic cancer cells treated for 72 hours. B) Tumor cell migration in ROCK1 siRNA treated cells. C) Fluorescence microscopic analysis of fasudil treated, co-cultured pancreatic cancer cells and cancer-associated fibroblasts. Cells were treated with fasudil for 48 hours and then were stained for α-SMA (red), Collagen I (green), and DNA (blue). D) Fluorescence microscopic analysis of fasudil treated CAFs. E) Fasudil treated, mono- and co-cultured pancreatic cancer cells and cancer-associated fibroblasts were harvested and analyzed by immunoblotting (dot blot) for Collagen I expression under non-denaturing conditions.

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Fig 4.

Effects of fasudil treatment on tumor stroma in KPC mice.

A) Pancreatic tumor tissues from vehicle, gemcitabine, or combination of gemcitabine plus fasudil treated KPC mice were harvested and stained for various stromal markers. Representative images are shown of H&E staining, α-SMA, Desmin, CD31, Collagen I, and Movat's pentachrome staining.

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Fig 5.

Effects of gemcitabine and fasudil treatment in the KPC model for PDAC.

A) Pancreatic tissues harvested and stained for CD31 were analyzed for positive staining per 20x field of view. B) Mouse liver sections were cut at multiple depths to assess the presence or absence of metastatic lesions in vehicle, gemcitabine, and gemcitabine plus fasudil treated tissues. Fasudil also enhanced the tumor growth inhibitory activity of gemcitabine in KPC mice. Three-dimensional volume measurements were acquired by ultrasonography of tumor bearing KPC mice before and after one (C) or two (D) 12-day treatment cycles. Percentage change is shown from baseline. IHC analysis of the proliferation marker Ki67 (E) and apoptosis marker cleaved-caspase 3 (CC3) (F) in epithelial tumor cells are also shown for mice treated in the various treatment cohorts. Fasudil enhanced survival of tumor bearing KPC mice. G) Kaplan-Meier curves of KPC mice treated with vehicle, gemcitabine, or the combination of gemcitabine plus fasudil. The combination treatment yields a significant improvement in overall survival compared to gemcitabine only group (Log-rank P value = 0.038). H) Pancreatic tissue from mice treated for three days with either vehicle or fasudil prior to a gemcitabine injection were analyzed for gemcitabine monophosphate concentrations in the tumor tissues. * P < 0.05. ** P < 0.01.

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