Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Summary

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Abstract

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

Introduction

The overall goal of this method is to develop a new breast cancer modeling approach based on an intraductal injection of Ad-Cre into the mouse MG. The Cre/loxP recombination-based genetic approach has been widely used to model human breast cancer in mice. The first generation of Cre/loxP-based breast cancer mouse models are generated by using Cre-expressing transgenic mice under the control of MEC-specific promoters (e.g., MMTV-Cre for luminal MECs and a portion of basal MECs, Wap-Cre and Blg-Cre for luminal progenitors and alveolar luminal MECs, K14-Cre for basal and a portion of luminal MECs^1,

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women’s Hospital.

1. Generation and maintenance of floxed mice

1. Obtain breast cancer-relevant floxed conditional knockout (e.g., Trp53^tm1Brn [referred to as Trp53^L/L], Brca1^tm1Aash [Brca1^L/L]) or conditional knock-in mouse lines (e.g., Gt(ROSA)26Sor^{tm1(Pik3ca*H1047R)Egan}) from The Jackson Laboratory (JAX) or NCI Mouse Models of Human Cancer Consortium (MMHCC) repository. In addition, to facilitate the chasin....

Results

Representative PCR genotyping results for the R26Y and Trp53^L alleles are shown in Figure 1.

Although, in principle, all 10 MGs can be subjected to the intraductal injection procedure, practically, the two fourth inguinal MGs are typically selected for injection, due to their easier accessibility and larger MG sizes (Figure 2). During the su.......

Discussion

The success of this approach for inducing mammary tumors from different subpopulations of MECs relies not only on choosing appropriate cell-type-specific promoters (to drive Cre expression) but also on the intraductal injection procedure itself. The idea behind this approach is that the injected Ad-Cre viruses are retained in the ductal tree, which is a concealed structure with lumen, and therefore, only MECs are exposed to the viruses and are infected by Ad-Cre. Due to the limited lumen space within the mammary ducts, i.......

Materials

Name	Company	Catalog Number	Comments
33-gauge needle	Hamilton	7803-05	point style 3 blunt
7mm Reflex Clip	Braintree Scientific	RF7 CS
Adenovirus, Ad-K5-Cre	University of Iowa Viral Vector Core	Ad5-bk5-Cre (VVC-Berns-1547)
Adenovirus, Ad-K8-Cre	University of Iowa Viral Vector Core	Ad5mK8-nlsCre
Alcohol	Fisher	HC800-1GAL	Prepare to 70% in use
biotinylated CD31	eBiosciences	13-0311-85
biotinylated CD45	eBiosciences	13-0451-85
biotinylated TER119	eBiosciences	13-5921-85
Bromophenol Blue	Sigma-Aldrich	B0126-25G
CD24-AF-700	BD Pharmingen	564237
CD24-PE	eBiosciences	12-0242-83
CD29-APC	eBiosciences	17-0291-82
CD29-PE	eBiosciences	12-0291-82
Hair Remover Lotion	Nair		9 Oz
Hamilton syringe	Hamilton	7636-01	0.025 mL
Iodophors	Betadine		10% Povidone-iodine
Isoflurane	Baxter	NDC 10019-360-40	1-2.5%
Loxicam	Norbrook	NDC 55529-040-10	5 mg/ml
Lubricant Eye Ointment	Akorn	NDC 17478-062-35
Micro-dissecting scissors	Pentair	9M	Watchmaker's Forceps
Micro-dissecting tweezers	Dumont	M5
Taq 5X Master Mix	New England Biolabs	M0285L