Fig 1: Ctbp2 drives tumor growth and metastasis in an orthotopic PDAC allograft mouse model.A Lysates of parental CKP and CKP cells with homozygous CRISPR/Cas9-based deletion of Ctbp2 (CKP/KO) immunoblotted with anti-Ctbp2 and GAPDH (loading control) antibodies. B Bioluminescent images obtained on the indicated days after orthotopic pancreatic implantation of luciferase-expressing CKP-luc or CKP/KO-luc cells into NSG mice. C Quantification of mean photon flux per second corresponding to images of mice in (B) using Living Image software (Perkin Elmer). D Total weight of pancreata of the mice in (B) at necropsy on day 21. E Kaplan–Meier survival analysis of mice with orthotopically implanted CKP-luc or CKP/KO-luc cells that were observed until humane endpoint. F Ascitic fluid volume of the mice in (B) at necropsy on day 21. (n = 5/group). *p < 0.05 and ***p < 0.001. p Values were obtained by performing two-tailed paired t-test between the two groups. Error bars indicate ±1.0 SD. Methods: (CKP PDAC cell line) The mouse CKP PDAC cell line was developed from the pancreas of a two-month-old CKP mouse according to the procedure described in [7]. The CKP cell line was verified as derived from PDAC tumor cells by showing elevated expression of PDAC markers Muc4 and Krt19 in the CKP cells vs. cells derived from normal mouse pancreata (Fig. S1B). CKP and all derivative cell lines were confirmed mycoplasma-free using a PCR-based assay (Cat No: ab289834; Abcam, Waltham, MA, USA). (CRISPR/Cas9-deletion of Ctbp2) We employed CRISPR-Cas9 gene editing to knock out Ctbp2 in CKP cells, and the resulting clones were validated for deletion of Ctbp2 by PCR of the Ctbp2 chromosomal locus and Ctbp2 immunoblotting (see Fig. 3 Methods). One of the several clones generated (CKP/KO) was used for all subsequent experiments. CKP and CKP/KO cell lines were grown in monolayers using RPMI media (Thermo Fisher, Waltham, MA, USA) supplemented with 4% FBS (Corning Life Sciences, Durham, NC, USA) and 1% penicillin–streptomycin (Thermo Fisher) at 37 °C in 5% CO2. DNA encoding CRISPR sgRNA targeting exon 3 of mCtbp2 was cloned into CRISPR OFP Nuclease Vector (Thermo-Fisher) using DNA oligonucleotides CP01 and CP02 (Table S2). mCtbp2 CRISPR plasmid-transfected CKP cells were sorted by flow cytometry 72 h post-transfection (λexc = 488 nm). Cells with highest Orange Fluorescent Protein (OFP) expression were pooled and cultured for a week until OFP expression was completely diminished. These cells were re-sorted for absence of OFP, single cell seeded in 96 well plates and grown to confluence. The clones were replica-plated for screening by western blotting. Further confirmation of homozygous knockout was done by PCR-amplification of the flanking regions of the indels with oligonucleotides DP331 and DP332 (Table S2) followed by cloning into pGEM-T Vector (Promega, Madison, WI, USA). Clones were sequenced to identify mutations in the mCtbp2 allele. (Luciferase expression in CKP and CKP/KO cells) The lentiviral vector pFULT expressing luciferase linked to Tomato under the control of the human pPGK1 promoter was purchased from Northwestern University (Gene Editing Transduction & Nanotechnology Core). CKP and CKP/KO cells were infected with this virus and 24 h later, the media in the cell culture dish was replaced with fresh media. After 24 h, lentiviral-infected cells were selected with puromycin (1.25 µg/ml) for 7 days, after which we performed an in vitro luciferase assay to confirm luciferase expression. All cells in the control plate died due to puromycin treatment. (Orthotopic injection of PDAC cells) We injected approximately 8 × 105 luciferase-expressing CKP-luc and CKP/KO-luc cells in PBS mixed with an equal volume of Cultrex Basement Membrane Matrix type 3 (R&D Systems, Minneapolis, MN, USA) into the pancreatic tail of 4-month-old male NSG mice (5 mice/cell line) as described [26–28]. NSG mice were procured from the Virginia Commonwealth University (VCU) Cancer Mouse Models Core Laboratory, and all animal studies were approved by the VCU Institutional Animal Care and Use Committee. The adherence of the implanted cells to the pancreas was measured immediately after orthotopic implantation by bioluminescent imaging in the IVIS Spectrum imager (Perkin-Elmer, Waltham, MA, USA) performed 10 min after subcutaneous injection of D-luciferin (150 mg/kg). Tumor growth was monitored similarly on days 7, 14, and 21 after implantation, via bioluminescent imaging after injection of the mice with D-luciferin.