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.

Keywords: CRISPR/Cas9; breast cancer; intraductal injection; invasive lobular carcinoma; mouse models; somatic gene editing.

Figure 1.

Intraductal injection of Lenti-Cre allows in vivo recombination in the mammary epithelium. (A) Fluorescence microscopy of GFP expression in a representative whole-mount mammary gland after intraductal injection of Lenti-GFP in FVB wild-type animals. n = 8. Mice were analyzed 14 d after injection. Bar, 5 mm. (B) Fluorescence microscopy of GFP expression in a representative whole-mount mammary gland after intraductal injection of Lenti-Cre in mT/mG Cre reporter mice. n = 8. Mice were analyzed 14 d after injection. Bar, 5 mm. (C) Immunohistochemical detection of GFP expression in a representative mammary gland section from mT/mG Cre reporter mice intraductally injected with Lenti-Cre. Bar, 100 µm. (D) Immunofluorescence analysis of mammary gland sections from Lenti-Cre-injected mT/mG Cre reporter mice showing GFP expression in cytokeratin 8 (CK8)-positive and CK14-positive cells. Bar, 25 µm.

Figure 2.

Intraductal injection of Lenti-Cre in Cdh1^F/F;Akt-E17K and Cdh1^F/F;Pten^F/F mice results in ILC formation. (A) Breeding strategy for a matched comparison of ILC formation induced by transgenic WapCre expression or Lenti-Cre injection in Cdh1^F/F;Akt-E17K mice. (B) In vivo bioluminescence imaging of luciferase expression in WapCre;Cdh1^F/F;Akt-E17K animals at 6 wk of age. (C) In vivo bioluminescence imaging of luciferase expression in Cdh1^F/F;Akt-E17K mice 24 wk after intraductal injection of Lenti-Cre. (D) Immunohistochemical analysis of E-cadherin, CK8, and phospho-AKT^Ser473 expression in WapCre;Cdh1^F/F;Akt-E17K (n = 15) tumors. Tumors were analyzed at 6 wk of age. Bars, 100 µm. (E) Immunohistochemical analysis of E-cadherin, CK8, and phospho-AKT^Ser473 expression in tumor sections from Lenti-Cre-injected Cdh1^F/F;Akt-E17K animals. n = 7. The tumor was analyzed 12 wk after injection. Bars, 100 µm. (F) Immunohistochemistry of E-cadherin, CK8, and phospho-AKT^Ser473 in tumor sections from Lenti-Cre-injected Cdh1^F/F;Pten^F/F animals. n = 8. Tumors were analyzed 14 wk after injection. Bars, 100 µm.

Figure 3.

Intraductal injection of pSECC-sgPten in Cdh1^F/F mice results in non-ILC tumors with strong immune infiltrations. (A) Analysis of pSECC-sgPten transduced Cre reporter cells 5 d after transduction. Percentage of GFP-positive cells and the spectrum of insertions/deletions (indels) of the targeted Pten alleles are shown by FACS and TIDE (tracking of indels by decomposition) analysis, respectively. The fraction of unmodified alleles is depicted in pink, while red bars (P < 0.001) and black bars (P > 0.001) represent the fractions of modified alleles. (B) H&E staining of a representative tumor section from Cdh1^F/F;Akt-E17K mice injected with pSECC-sgNT. n = 4. Tumors were analyzed 16 wk after injection. Bar, 100 µm. (C) Immunohistochemical detection of E-cadherin-, CK8-, CD4-, CD8-, and B220-expressing cells in tumor sections from Cdh1^F/F mice injected with pSECC-sgPten. Tumor lesions were analyzed 25 wk after injection. Bars, 100 µm. (D) Histological classification of tumors from Cdh1^F/F mice injected with pSECC-sgPten. n = 48. ILC-like classification refers to lesions that are too small to display the typical ILC histological phenotype with the characteristic growth pattern. (E) TIDE analysis of the targeted Pten alleles in a representative tumor from pSECC-sgPten-injected Cdh1^F/F mice.

Figure 4.

ILC formation in WapCre;Cdh1^F/F;Cas9 mice injected with LentiGuide-sgPten. (A) Expression of Cas9 from the Cre conditional Cas9 allele following in vitro transduction of Cdh1^F/F;Cas9 MMECs with Adeno-Cre, as visualized by immunoblotting with anti-Flag and anti-Cas9 antibodies. Pol II is shown as loading control. (B) TIDE analysis of the targeted Pten alleles in Cdh1^F/F;Cas9 MMECs cotransduced with Lenti-Cre and LentiGuide-sgPten. (C) Immunohistochemical detection of E-cadherin-, CK8-, CD4-, CD8-, and B220-expressing cells in tumor sections from WapCre;Cdh1^F/F;Cas9 mice injected with LentiGuide-sgPten. Tumor lesions were analyzed 25 wk after injection. Bars, 100 µm. (D) Histological classification of tumors from WapCre;Cdh1^F/F;Cas9 mice injected with LentiGuide-sgPten. n = 27. (E) TIDE analysis of the targeted Pten alleles in a representative tumor from LentiGuide-sgPten-injected WapCre;Cdh1^F/F;Cas9^F/F mice. (F) Representative immunofluorescence imaging of tumor sections from LentiGuide-sgPten-injected WapCre;Cdh1^F/F;Cas9 mice, stained with antibodies against CK8, PTEN, and E-cadherin. Bar, 25 µm.