Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Abstract

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of αβ and γδ T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

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

“…By using high-titer lentiviral vector preparations, we achieved extensive transduction of the ductal system and in vivo Cre-mediated recombination when using Lenti-Cre preparations in mT/mG Cre reporter mice. This recapitulates previous studies using adenoviral vectors for somatic Cre delivery to murine mammary tissue (Rutkowski et al 2014;Tao et al 2014). We observed that the target cell population was composed of both CK8-positive luminal epithelial cells and CK14-positive basal cells.…”

“…S1C,D). These data demonstrate that intraductal injection of Cre-encoding lentiviruses induces efficient in vivo recombination of conditional alleles in mammary epithelium, as previously shown by other groups using Adeno-Cre (Russell et al 2003;Rutkowski et al 2014;Tao et al 2014).…”

“…In our study, we set out to implement intraductal injections of lentiviral vectors as a tool to achieve mammary gland-specific Cre expression and/ or CRISPR/Cas9-mediated genome editing. In the past, intraductal injection of Cre-encoding adenoviruses was successfully used to activate expression of oncogenic fusion genes in the mammary gland of genetically engineered mice, leading to mammary tumors (Rutkowski et al 2014;Tao et al 2014). To verify the applicability of this technique for intraductal delivery of lentiviruses, we performed injections of female virgin FVB mice with a lentiviral vector expressing GFP (n = 8), revealing efficient transduction of the ductal tree ( Fig.…”

IL15 Agonists Overcome the Immunosuppressive Effects of MEK Inhibitors

“…To investigate the therapeutic potential of combining trametinib and ALT-803 against established KRas -mutated tumors, we utilized a syngeneic tumor model derived from an autochthonous breast cancer initiated in triple transgenic (L-Stop-L- KRas G12D p53 flx/flx L-Stop-L- Myristoylated p110α ) mice with adenovirus-Cre (11, 12, 33). We chose this cell line, termed br east- p 53- K Ras- p110 alpha (Brpkp110), to model treatment against tumors that evade single molecule targeting of the MAPK pathway through PI3K activation, as has been commonly reported in human cancer cells (34–36).…”

“…The Brpkp110 primary mammary tumor cell line was generated by culturing a mechanically dissociated C57BL/6 L-Stop-L- Kras G12D p53 flx/flx L-Stop-L- Myristoylated p110α − GFP flx/+ primary breast tumor mass as previously described (15). Tumor cells were passaged a total of 10 times and tested for mycoplasma before deriving the Brpkp110 cell line.…”