S. Liu1, J. Yu1, R. Sanchez1, E. Rozengurt2, F. Brunicardi1 1David Geffen School Of Medicine, University Of California At Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2David Geffen School Of Medicine, University Of California At Los Angeles,Division Of Digestive Disease,Los Angeles, CA, USA
Introduction: Recent advances in CRISPR-Cas9 gene editing and organoid culturing are leading to pancreatic cancer (PDAC) tumor models with unprecedented speed and precision by introducing driver mutations such as KrasG12D, P53 mutation, which will hopefully identify early detection PDAC biomarkers. Over-expression of BIRC5 has been found in early stage of PDAC in human specimens and is a promising candidate for an early detection biomarker. The purpose of this study is to study BIRC5 as an early detection biomarker by taking the advantage of CRISPR-Cas9 and organoid techniques to develop an early onset pancreatic 3D tumor model.
Methods: Three-dimensional culture conditions were optimized to maintain mouse pancreatic ductal progenitor organoids (ORGs). sgRNA was well designed and cloned into a GFP-tagged lentiviral vector to generate two vectors: Lenti-sgKrasG12D-GFP and Lenti-sgKrasG12D-sgp53KO-GFP. Co-transfection was performed using Lenti-sgRNA-GFP and Lenti-Cas9-2A-tdTomato (tdT) to infect ORG. KrasG12D and p53 mutations were confirmed by genome DNA sequencing. BIRC5 expression in ORGs and human specimens was measured using western blot and immunofluorescence assay. Knockdown of Kras and overexpression of wild type p53 was carried out. A reporter assay using BIRC5-GLuc was performed in these ORGs.
Results:Immunofluorescence demonstrated that BIRC5 was expressed in PanIN3 and PDAC specimens. ORGs were established in matrigel with additional growing factors in culture media. GFP (green), tdT (red) or GFP/tdT double staining was observed in ORGs following co-infection. Tumoroid ORGs were observed in CRISPR-Cas9 engineered ORGs displaying GFP/tdT double staining in 3-D culturing with either GFP or tdT only (Fig). The expression of GFP and tdT were further confirmed in the cryosection and paraffin sections. KrasG12D mutation only resulted in very limited expression of BIRC5 in ORGs, however KrasG12D/p53KO mutations markedly increased BIRC5 expression. Knockdown of KrasG12D expression did not alter BIRC5 levels, however, restoration of wild type P53 expression resulted in significant reduction of BIRC5 levels in ORGs (p<0.05). Furthermore, BIRC5-GLuc reporter assay revealed GLuc expression only in the ORGs with both KRAS and P53 mutations.
Conclusion:CRISPR-Cas9 engineering of pancreatic ductal organoids with KRAS/P53 driver mutations resulted in a tumoroid ORGs associated with BIRC5 overexpression. BIRC5 was over-expressed in PanIN3 and PDAC. These data support the hypothesis that BIRC5 is biomarker for early detection of PDAC.
