Abstract 2687: Rapid in vivo testing of tumor suppressors in ILC by CRISPR-Cas9 mediated somatic gene editing of the mammary gland

Abstract

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype, accounting for 5% to 15% of breast tumors.The majority of ILCs are characterized by the complete loss of the cell adhesion protein E-cadherin encoded by the CDH1 gene. However, WAPcre;Cdh1F/F mice with mammary gland-specific E-cadherin loss do not develop ILC, unless coupled with the additional disruption of a tumor suppressor gene, like Pten or Trp53. Compound mutant mice develop lesions that closely resemble the human disease in terms of histology and invasivity. However, genome-wide sequencing projects and forward genetic screens identified a number of additional putative ILC-initiating loci. Hence, there is now an urgent need for validation and characterization of these candidate cancer genes. We sought to determine if it was possible to deploy CRISPR/Cas9 technology to somatically inactivate candidate tumor suppressor genes in mammary gland tissue of adult mice. For this purpose we performed in Cdh1F/F mice intraductal injections of high-titer lentiviral vectors carrying different combinations of Cre, Cas9 and sgRNAs targeting several candidate genes, including Pten, Tgfbr2, Myh9, Nf1 and Fbxw7. We found that Cas9-bearing vectors elicited strong immune responses against transduced cells, which resulted in small ILC lesions surrounded by immune cells. However, when Cas9 was expressed endogenously in the mammary tissue from a knock-in allele, intraductal injection of lentiviral vectors encoding the sgRNA moiety of the CRISPR system was sufficient to induce penetrant and multifocal ILC formation in WAPcre;Cdh1F/F;Cas9 female mice. Sequencing revealed specific mutations clustered in the CRISPR targeted gene, and positive selection for loss-of-function indels. Indeed, immunohistochemistry and immunofluorescence analysis confirmed that tumors were largely deficient for E-cadherin and the targeted gene. In sum, we have successfully established a new platform for in vivo CRISPR-Cas9 mediated somatic gene editing in the mouse mammary gland. This rapid and versatile platform can be used to identify novel tumor drivers (e.g.by employing forward genetic screens) and validate candidate cancer genes in ILC and other breast cancer types. Citation Format: Stefano Annunziato, Sjors Kas, Micha Nethe, Hatice Yucel, Jessica del Bravo, Colin Pritchard, Rahmen Bin Ali, Bas van Gerwen, Bjorn Siteur, Anne Paulien Drenth, Eva Schut-Kregel, Sjoerd Klarenbeek, Ivo Huijbers, Martine van Miltenburg, Jos Jonkers. Rapid in vivo testing of tumor suppressors in ILC by CRISPR-Cas9 mediated somatic gene editing of the mammary gland. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2687.

In Vivo Modeling of Tumor Heterogeneity for Immuno‐Oncology Studies: Failures, Improvements, and Hopes

“…Likewise, to avoid Cas9-associated immunogenicity in vivo, tumor cell lines can be transiently exposed to Cas9 expression through either non-integrating viruses or inducible control of Cas9 expression (Dow et al, 2015). Alternatively, Cas9-transgenic mice can be used as recipients for tumor cell lines transduced with sgRNAs only or lines that constitutively express Cas9 because the mice are tolerized to Cas9 (Annunziato et al, 2016). Additionally, the selective CRISPR antigen removal (SCAR) system was created to prevent immune-mediated rejection of CRISPR-modified tumor cells and facilitate in vivo high-throughput genetic screens by removing Cas9 and other immunogenic vector components after genome editing (Dubrot et al, 2021).…”