Fig. 6

CRO-67 treatment significantly decreased αSMA⁺ CAF frequency, and increased the fraction of opened blood vessels and low-density collagen fibrils in PDAC orthotopic mouse tumors. (a) Schematic representation of orthotopic PDAC mouse model made with Biorender. 7 weeks old, female Balb/c nude mice were injected with luciferase-expressing MiaPaCa-2 cells and immortalized human primary CAFs at a 1:1 ratio of cells (10⁶ each) in the pancreas. 4 weeks post-surgery, mice were randomized into the vehicle control or CRO-67 treatment group (10 mice/group), based on (b) mouse tumor volume via ultrasound measurements. Mice were treated with Vehicle Control (90% saline + 10% DMSO) or CRO-67 (60 mg/kg), twice daily via intraperitoneal injection (i.p.), for 5 weeks. At endpoint, mouse pancreatic tumors were harvested, fixed in 4% paraformaldehyde, for tissue processing and subsequent immunohistochemistry analyses. Ex vivo organ luminescence for metastasis detection was performed via bioluminescence imaging using IVIS SpectrumCT. Individual tumor volume was measured by ultrasound as a percentage of start and number of metastatic sites per mouse with the percentage of mice presenting any metastasis were quantified for (c) all tumors and for (d) tumors with > 150% growth at endpoint compared to start. (e) Representative images and quantification of α-smooth muscle actin (αSMA) positive cells. (f) Representative images and quantification of CD31 positive cells. (g) Representative bright field images showing picrosirius red stained tumor sections and (h) representative images of picrosirius red stained imaged using polarized light. Quantification of total picrosirius red coverage (i) and low–high birefrigent collagen (j–l) are shown in the graphs. Quantification of each stain was performed using MATLAB programming. Symbols show quantification of individual mice. Bars represent mean ± SEM. Asterisks indicate statistical significance as assessed by t-test (*p ≤ 0.05, **p ≤ 0.01).