Abstract 2586 Evaluating the effect of RNAi silencing of PTDSS1 in pancreatic cancer cells

Pericytes are mesenchymal cells that envelop microvessels and contribute to their stability through the secretion of collagen IV and other basement membrane components.They are found in every tissue type, however are difficult to identify via immunostaining because of their similarities to other mesenchymal cell types, like fibroblasts.The lack of cellular markers has contributed to the paucity of available pericyte cell models for study.Through a comprehensive cellular and molecular dissection of the human breast, our laboratory has developed a method for identifying, isolating, and culturing every cell type in the breast-including pericytes.We are focused on pericytes because, using our novel pericyte cell model, we have discovered that they induce rapid tumor cell proliferation that was even greater than isogenic fibroblast controls.Overcoming problems with markers, we have discovered the expression levels of CD36 and CD26 are reliable markers for pericytes and fibroblasts cultures, respectively.Thus, it is critical that primary cultures be monitored for these two markers to identify potential cross-contamination of the two cell types that are otherwise morphologically similar.To monitor potential changes in the expression of these markers in our primary cultures, we needed a method to quantify staining.Using the CellProfiler software package, I developed a facile pipeline to analyze cytoplasmic staining of CD36 and CD26 in primary pericyte and fibroblast cultures.We found CD36 staining was indeed dramatically different between these cultures, as staining was 163-fold higher in pericytes.CD26 was a different story.Although the average staining was indeed statistically higher in fibroblasts (p=4.97e-8),there was overlap, which makes quantification essential.This pipeline is versatile and can be easily adapted for other cell surface or cytoplasmic markers.Our approach offers a robust method for ensuring the integrity of primary pericyte cultures, facilitating downstream investigations into the role of these cells in tumorigenesis.