PTEN Deletion in Pancreatic Cancer Associated Fibroblasts Decreases Hydraulic Permeability Independent of Collagen Fiber Alignment in a 3D Microfluidic Model of the Tumor Stroma

Using a microfluidic 3D model of the tumor stroma, this study set out to characterize how loss of the tumor suppressor Phosphatase and Tensin Homolog (PTEN) in pancreatic cancer associated fibroblasts(CAFs) alters two biophysical properties of the tumor microenvironment: i) collagen fiber alignment and ii) hydraulic permeability. Aligned collagen fibers have been shown to provide contact guidance for tumor cells to metastasize while hydraulic permeability is an indicator of how well flow and drugs can penetrate into the tumor and thus provide important parameters to profile the effects of CAFs in pancreatic cancer. We hypothesized that loss of PTEN in CAFs would result in more aligned collagen fibers and decreased hydraulic permeability, characteristic of aggressive pancreatic tumors. The microfluidic model consisted of polydimethylsiloxane devices fabricated using soft lithography. The devices consisted of a straight channel (5mmx500umx1mm) with 4mm ports. CAFs, isolated from human pancreatic tumors, were suspended in a rat tail type I collagen hydrogel, injected into the channel, and cultured between 2–4 days prior to measurements. Collagen fiber alignment was assessed by taking confocal reflectance images of the collagen fibers within the device and computing an alignment index (a ratio between the frequency of the most aligned region of the image and the ideally random case). With this criteria, alignment indices closer to 1 indicate random alignment while higher values indicate more aligned collagen fibers. The hydraulic permeability was determined by flowing a rhodamine‐bovine serum albumin dye through the channel, measuring the average fluid velocity through the collagen type I hydrogel, and using Darcy's Law to calculate the hydraulic permeability. The conditions tested were control pancreatic CAFs (shnc), pancreatic CAFs with PTEN silenced with shRNA (shPTEN), and acellular collagen Type I hydrogel. We detected no significant differences in collagen fiber alignment when comparing shnc, shPTEN, and acellular collagen conditions. In contrast, the hydraulic permeability decreased significantly for the shPTEN condition when compared to both shnc and acellular collagen conditions. These results indicate that loss of PTEN in CAFs results in decreased hydraulic permeability of the tumor stroma that is independent of collagen fiber alignment. Furthermore, the decrease in the hydraulic permeability conferred by shPTEN CAFs can explain why loss of PTEN in pancreatic tumors results in resistance to therapy since convective delivery of therapeutic agents into the tumor is impeded. Since fiber alignment was not observed to be impacted by expression of PTEN in CAFs, we suspect that this decrease in hydraulic permeability is mediated by the deposition of extracellular matrix molecules that increase the hydraulic resistance of the tumor stroma. Support or Funding Information Funding to JWS from: American Cancer Society (IRG‐67‐003‐50), American Heart Association (15SDG25480000), and Ohio State University Institute for Materials Research. CE and JWS acknowledge support from Pelotonia. 1Collagen fiber alignment index quantification. No differences were detected between acellular, shnc, and shPTEN collagen using ANOVA (F>0.05). Error bars indicate the standard error of the mean.2Hydraulic permeability decreases significantly due to PTEN deletion in CAFs. Statistical testing was done using ANOVA with Tukey's HSD post testing. (n = 4–8 across all conditions, α set to 0.05 for statistic significance). Error bars indicate the standard error of the mean.