Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.

Preclinical in vivo validation of target genes for therapeutic intervention requires careful selection and characterization of the most suitable animal model in order to assess the role of these genes in a particular process or disease. To this end, genetically engineered mouse models (GEMMs) are typically used. However, the appropriate engineering of these models is often cumbersome and time consuming. Recently, we and others described a modular approach for fast-track modification of existing GEMMs by re-derivation of embryonic stem cells (ESCs) that can be modified by recombinase-mediated transgene insertion and subsequently used for the production of chimeric mice. This 'GEMM-ESC strategy' allows for rapid in vivo analysis of gene function in the chimeras and their offspring. Moreover, this strategy is compatible with CRISPR/Cas9-mediated genome editing. This protocol describes when and how to use the GEMM-ESC strategy effectively, and it provides a detailed procedure for re-deriving and manipulating GEMM-ESCs under feeder- and serum-free conditions. This strategy produces transgenic mice with the desired complex genotype faster than traditional methods: generation of validated GEMM-ESC clones for controlled transgene integration takes 9-12 months, and recombinase-mediated transgene integration and chimeric cohort production takes 2-3 months. The protocol requires skills in embryology, stem cell biology and molecular biology, and it is ideally performed within, or in close collaboration with, a transgenic facility.

The hepatocyte growth factor (HGF) is a pleiotropic cytokine that influences mitogenesis, motility and differentiation of many different cell types by its tyrosine kinase receptor c-Met. We previously demonstrated that the c-Met/HGF system is present and functionally active during postnatal testis development. We found also that spermatozoa express c-Met and that HGF has a positive effect on the maintenance of sperm motility. In the present paper, we extend our study on the germ cells at different stages of differentiation during the postnatal development of the testis. We demonstrate that c-met is present in rat spermatogonia, pachytene spermatocytes and round spermatids and that HGF significantly increases spermatogonial proliferation in 8-to 10-day-old prepubertal rats. At this age HGF does not affect Sertoli cells and peritubular myoid cells proliferation. In addition, we studied the effect of the factor on germ cell apoptosis and we show that HGF prevents the germ cell apoptotic process. We also studied the effect of HGF on 18-to 20-day-old and 28-to 30-day-old rat testes. At these ages also the factor significantly increases germ cell duplication and decreases the number of apoptotic cells. However, the effect on programmed cell death is higher in the 8-to 10-day-old rats and declines in the older animals.In conclusion, we report that rat germ cells (spermatogonia, pachytene spermatocytes and round spermatids) express c-met and that HGF modulates germ cell proliferating activity and apoptosis in vitro. These data indicate that the c-Met/HGF system is involved in male germ cell homeostasis and, consequently, has a role in male fertility.

Hepatocyte growth factor (HGF) regulates many cellular functions acting through c-Met, its specific tyrosine kinase receptor. We previously reported that in prepuberal rats HGF is secreted by the peritubular myoid cells during the entire postnatal testicular development and by the Sertoli cells only at puberty. We have also demonstrated that germ cells at different stages of development express c-Met and that HGF modulates germ cell proliferation and apoptosis. In the present article, we extend our study to the interstitial compartment of the testis and demonstrate that the c-Met protein is present on Leydig cells. The receptor is functionally active as demonstrated by the detected effects of HGF. We report in this article that HGF significantly increases the amount of testosterone secreted by the Leydig cells and decreases the number of Leydig cells undergoing apoptosis. The antiapoptotic effect of HGF is mediated by caspase-3 activity because the amount of the active fragment of the enzyme is decreased in Leydig cells cultured in the presence of HGF. However, treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. These data indicate that HGF regulates the functional activities of Leydig cells. Interestingly, the steroidogenetic activity of the cells is increased by HGF in cultured explants of testicular tissues as well as the antiapoptotic effect of HGF. Therefore, our data indicate that HGF has a crucial role in the regulation of male fertility.

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.