Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland

Abstract

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.

Genetic Dissection of Cancer Development, Therapy Response, and Resistance in Mouse Models of Breast Cancer

“…We have shown that intraductally injected lentiviruses can target tumor-initiating cells of both the basal and the luminal compartment, allowing modeling of both ILC and basallike tumors in mice with the corresponding set of relevant predisposing alleles. For example, intraductal injection of Cre-encoding lentiviruses in Cdh1 F/F ;Pten F/F mice induced the formation of ILCs that were undistinguishable from the ILCs arising in the original WAPcre;Cdh1 F/F ; Pten F/F model (Annunziato et al 2016). Somatic Cre delivery may more accurately recapitulate sporadic tumor initiation by allowing titratable and spatiotemporally controlled delivery of viruses to mammary tissue.…”

“…3). Regarding CRISPR-based in vivo editing approaches, we and others have shown that somatic delivery of the bacterial Cas9 protein has the considerable drawback of eliciting strong and specific immune responses in immunocompetent animals (Wang et al 2015;Annunziato et al 2016). This problem can be overcome by employing knock-in models that are tolerant to Cas9 because of constitutive or conditional expression of Cas9 or catalytically inactive dCas9-effector fusions (which allow for transcriptional silencing/activation of endogenous alleles) (Platt et al 2014;Sánchez-Rivera and Jacks 2015).…”

“…This problem can be overcome by employing knock-in models that are tolerant to Cas9 because of constitutive or conditional expression of Cas9 or catalytically inactive dCas9-effector fusions (which allow for transcriptional silencing/activation of endogenous alleles) (Platt et al 2014;Sánchez-Rivera and Jacks 2015). We have recently reported somatic induction of oncogenic loss-of-function mutations in mice with mammary-specific expression of Cas9 by intraductal injection of single-guide RNA (sgRNA)-encoding lentiviruses, which eventually led to ILC formation (Annunziato et al 2016).…”

Somatic Genome Editing Goes Viral

“…A new study published by Jonkers and colleagues in Genes & Development [1] provides a timely example of how CRISPR has spawned a new in vivo toolkit equipped to deal with the need for increasingly complex genetic mouse models. Using a viral-based approach, Annunziato et al show that Cas9-mediated genome editing can produce genetically and histologically accurate invasive breast cancers in mice and thus delineate a path forward to putatively define genetic drivers of this disease and develop tailored preclinical model systems.…”

An optimized toolkit for precision base editing

“…As expected, tumor nodules showed efficient editing of the target Ctnnb1 locus, to create activating S45F mutations (Figure 3h). An alternate approach to in vivo somatic base editing is the generation of temporally regulated transgenic strains, which enables the manipulation of tissues and cell types that cannot be easily transfected in vivo, and avoids the potential immunogenicity of exogenous Cas9 delivery 21,22 . We generated knock-in transgenic mouse embryonic stem cells (ESCs), in which BE3 or RA expression is controlled by the TRE promoter.…”