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CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout Published OnlineFirst August 16, 2016; DOI: 10.1158/0008-5472.CAN-16-1272 Cancer Tumor and Stem Cell Biology Research CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma Josephine Walton1,2, Julianna Blagih3, Darren Ennis1, Elaine Leung1, Suzanne Dowson1, Malcolm Farquharson1, Laura A. Tookman4, Clare Orange5, Dimitris Athineos3, Susan Mason3, David Stevenson3, Karen Blyth3, Douglas Strathdee3, Frances R. Balkwill2, Karen Vousden3, Michelle Lockley4, and Iain A. McNeish1,4 Abstract – – There is a need for transplantable murine models of ovarian ating novel ID8 derivatives that harbored single (Trp53 / )or – – – – high-grade serous carcinoma (HGSC) with regard to mutations in double (Trp53 / ;Brca2 / ) suppressor gene deletions. In these the human disease to assist investigations of the relationships mutants, loss of p53 alone was sufficient to increase the growth between tumor genotype, chemotherapy response, and immune rate of orthotopic tumors with significant effects observed on the microenvironment. In addressing this need, we performed whole- immune microenvironment. Specifically, p53 loss increased exome sequencing of ID8, the most widely used transplantable expression of the myeloid attractant CCL2 and promoted the model of ovarian cancer, covering 194,000 exomes at a mean infiltration of immunosuppressive myeloid cell populations into – – – – depth of 400Â with 90% exons sequenced >50Â. We found no primary tumors and their ascites. In Trp53 / ;Brca2 / mutant functional mutations in genes characteristic of HGSC (Trp53, cells, we documented a relative increase in sensitivity to the PARP Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally inhibitor rucaparib and slower orthotopic tumor growth – – active. Homologous recombination in ID8 remained intact in compared with Trp53 / cells, with an appearance of intratumoral þ functional assays. Further, we found no mutations typical of clear tertiary lymphoid structures rich in CD3 T cells. This work cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma validates new CRISPR-generated models of HGSC to investigate (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. its biology and promote mechanism-based therapeutics disco- Using CRISPR/Cas9 gene editing, we modeled HGSC by gener- very. Cancer Res; 76(20); 1–12. Ó2016 AACR. Introduction HGSC through a variety of additional mechanisms, including somatic BRCA1/2 mutation and epigenetic loss of BRCA1 expres- Ovarian high-grade serous carcinoma (HGSC) is the common- sion (4). Another key development has been the finding that est subtype of human ovarian cancer (1), and long-term survival HGSC may arise in the fimbriae of the fallopian tube, rather than remains poor (2). Mutation in TP53 is essentially universal (3, 4), the ovarian surface epithelium (OSE; ref. 7). Early serous tubal whereas germline mutation in BRCA1 or BRCA2 is observed in intra-epithelial carcinoma (STIC) lesions in the tubal fimbriae are approximately 15% cases (5, 6). These mutations impair the observed in up to 10% of women with germline BRCA1/2 muta- ability to repair DNA double-strand breaks (DSB) via homolo- tions undergoing prophylactic salpingo-oophorectomy (8). gous recombination (HR). The Cancer Genome Atlas consortium Although a tubal origin of HGSC is widely accepted (9), STIC suggested that HR defects may be present in approximately 50% lesions are only seen in around half of HGSC cases (10). Recent data from mouse models (11, 12) and clinical HGSC samples 1Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, (13) suggest that the ovary itself may still be crucial in the University of Glasgow, Glasgow, United Kingdom. 2Centre for Cancer pathogenesis of HGSC, and it is possible that there could be and Inflammation, Barts Cancer Institute, Queen Mary University of more than one origin of this tumor. London, London, United Kingdom. 3Cancer Research UK Beatson Institute, Glasgow, United Kingdom. 4Centre for Molecular Oncology, The absence of reliable murine models has been a major Barts Cancer Institute, Queen Mary University of London, London, impediment in HGSC research (14). This is particularly impor- United Kingdom. 5Department of Pathology, Institute of Cancer tant for investigation of the immune microenvironment. The Sciences, University of Glasgow, Glasgow, United Kingdom. presence of tumor-infiltrating CD8 T lymphocytes (TIL) and Note: Supplementary data for this article are available at Cancer Research tertiary intraepithelial lymphoid aggregates are both associated Online (http://cancerres.aacrjournals.org/). with improved prognosis in HGSC (15, 16), whereas intratu- Corresponding Author: Iain A. McNeish, University of Glasgow, Wolfson Wohl moral immunosuppressive myeloid and lymphoid cells (17, Cancer Research Centre, Garscube Estate, Glasgow G61 1QH, United Kingdom. 18) are associated with poor prognosis. However, it is unclear Phone: 44-141-330-3968; Fax: 44-141-330-4127; E-mail: whetherorhowspecific genomic events in HGSC influence the [email protected] immune microenvironment. doi: 10.1158/0008-5472.CAN-16-1272 The ID8 model, first described in 2000 (19), remains the Ó2016 American Association for Cancer Research. only transplantable murine model of ovarian cancer routinely www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 16, 2016; DOI: 10.1158/0008-5472.CAN-16-1272 Walton et al. available. Whole ovaries from C57Bl/6 mice were trypsin- and plated onto 96-well plates (10 cells/mL). Single-cell colonies digested, and the dissociated cells passaged in vitro, initially in were expanded for DNA extraction, protein extraction, and the presence of EGF. After approximately 20 passages, cells lost cryopreservation. contact inhibition, and ten separate clones were derived, of which PCR primers spanning potential sites of deletion were designed ID8 is the most widely used. Following intraperitoneal injection (Trp53:F50-cttccctcacattcctttcttg-30;R50-gctgttaaagtagaccctgggc-30, of ID8 in syngeneic mice, diffuse peritoneal carcinomatosis, with Brca2:F50-catggagggagtcacctttg-30,R50-gctctggctgtctcgaactt-30). blood-stained ascites, develops in approximately 110 days (19). Clones with large PCR deletions were selected for subsequent Over 100 publications have utilized the ID8 model, but none has analysis. Remaining clones were screened using the Surveyor characterized it in light of current understanding of human Nuclease Assay (Integrated DNA Technology). Mutations were ovarian cancer biology. confirmed by Sanger sequencing. All sequence alignment was Here, we show that parental ID8 lacks mutations in Trp53, performed using MAFFT version 7 (http://mafft.cbrc.jp/). Brca1, and Brca2, and demonstrates HR competence in functional assays. We have used CRISPR/Cas9 gene editing technology to Immunoblot and cytokine array generate single (Trp53) and double (Trp53;Brca2) knockout deri- Note that 15 mg of total protein was electrophoresed at 140 V for vatives of ID8 and evaluated their utility as a model of human 1 hour, transferred onto nitrocellulose, and blocked in 5% nonfat HGSC. In particular, we show that loss of individual genes results milk. Antibodies used were as follows: p53 (CM5, Novacastra and in significant alterations in immune cell infiltration into the Ab26, Abcam) and b-actin (Sigma; A1978). Membranes were tumor microenvironment. exposed on a Chemi-doc MP (Biorad) with ECL (b actin) and ECL prime (p53). For array experiments, 5 mL supernatant was Materials and Methods collected from 106 cells plated for 16 hours on a 10-cm plate, and centrifuged (2,000 rpm for 5 minutes). Mouse Cytokine Antibody Cell culture Array C1 (C-series) was blotted according to the manufacturer's ID8 cells, obtained from Dr. Katherine Roby (University of instructions (RayBiotech, Inc.). Kansas Medical Center, Kansas City, KS), were cultured in DMEM supplemented with 4% fetal calf serum, 100 mg/mL penicillin, 100 mg/mL streptomycin, and ITS (5 mg/mL insulin, 5 mg/mL Quantitative reverse transcriptase PCR Â 5 transferrin, and 5 ng/mL sodium selenite). As ID8 was obtained RNA was extracted from 3 10 cells in log-growth phase, and directly from their original source, separate short tandem repeat 2 mg was reverse transcribed (Applied Biosystems High Capacity fi validation was not performed. For cytotoxicity assays, cells were Reverse Transcription Kit). cDNA (50 ng) was ampli ed using 10x plated onto 24 well plates (3 Â 103 cells/well) in triplicate. iTaq qRT-PCR master mix (Biorad), 20X primer probe mix in a total of 20 mL under the following cycle parameters: 2 minutes: Survival was assessed by MTT assay (Nutlin-3) or sulphorhoda- mine B assay (rucaparib) after 72 hours. 50 C, 10 minutes: 95 C, 40X (15 seconds: 95 C; 1 minute: 60 C). All qRT-PCR primers were purchased from Applied Biosystems and Trp53 custom made from Sigma Aldrich: F 50-catcacctcactg- Next-generation sequencing catggac-30,R50-cttcacttgggccttcaaaa-30, probe 50-ccccaggatgttgag- Whole-exome sequencing and analysis were performed by gagt-30. Values were normalized to Rpl-34. Beckman Coulter Genomics. Full details are given in Supplemen- tary Methods. Summary results are presented in Supplementary gH2AX/Rad51 assay fi Tables S1 to S3. Primary sequencing data (BAM and VCF les) are Cells were seeded on coverslips and treated with rucaparib (10 available in the ArrayExpress database (http://www.ebi.ac.uk/
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