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^1,2,3,4,5)^6,7,8,9. However, while these Cre transgenic lines enable spatial control of Cre expression (i.e., in different subsets of MECs), they do not allow temporal control of Cre expression and Cre/loxP-mediated genetic manipulation. The second generation of Cre/loxP-based breast cancer mouse models utilize inducible Cre activity/expression approaches (e.g., use of Cre-estrogen receptor fusion [CreER], which can only induce Cre/loxP recombination upon administration of tamoxifen), and as a result, these genetic tools permit both spatial and temporal controls of the activation of oncogenic events in MECs (e.g., K8-CreER- and K5-CreER-based models)^10,11,12. In both generations of breast cancer mouse models, as promoters used to drive Cre or CreER expression (e.g., Krt8, Krt5) may also be active in epithelial cells of other organs (i.e., they are cell-type-specific but not organ-specific) or have a leaky expression in cell types other than epithelial cells (e.g., MMTV, which has leaky activity in bone marrow hematopoietic cells), these approaches may lead to the development of unwanted cancer(s) in other organ(s). If these unexpected cancers cause lethality in the affected mice, the original purpose of modeling breast cancer in these mice may be prohibited (e.g., MMTV-Cre-driven oncogenic events may lead to hematopoietic malignancies and early death of the mice, due to leakiness of the MMTV promoter in hematopoietic cells)⁴.

Here we report a breast cancer modeling approach in mice that allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner. This approach is based on an intraductal injection of Ad-Cre into mouse MGs (and is, thus, organ-specific). Cre expression can be controlled by using different MEC subpopulation-specific promoters embedded in the adenoviral vector (e.g., Krt8 for luminal MECs, Krt5 for basal MECs, thus achieving cell-type specificity). Cancer induction in MGs can be temporally controlled by an injection of Ad-Cre into mice at different ages, starting from 3-4 weeks of age (pubertal) to the adult stage.

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 chasing of MECs that undergo Cre-mediated recombination, a conditional Cre-reporter line can also be obtained from JAX (e.g., Gt(ROSA)26Sor^tm1(EYFP)Cos [referred to as R26Y]).
2. Breed Trp53^L/L homozygous mice with R26Y homozygous reporter mice or with homozygous mice carrying the R26Y reporter alleles and any additional floxed conditional knockout or knock-in alleles for different mouse models, to obtain heterozygous F1 male and female progeny.
3. Intercross heterozygous F1 male and female mice to obtain F2 compound female mice that are homozygous for each allele (as experimental mice), as well as R26Y-only homozygous females (as control mice). Genotype F2 mice based on the PCR primers and cycling conditions listed below, by setting up two standard 20 µL PCR reactions (using Taq 5X Master Mix) in two different PCR tubes, one with the R26Y primers and the other with the Trp53^L primers. Use adult mice (typically around 2–4 months of age) for all breeding.
   1. For R26Y, perform PCR at 94 °C for 3 min, then at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min for 35 cycles, followed by 72 °C for 3 min, and maintaining at 14 °C. Use primers (i) R26YFP-1: AAA GTC GCT CTG AGT TGT TAT; (ii) R26YFP-2: GCG AAG AGT TTG TCC TCA ACC; (iii) R26YFP-3: GGA GCG GGA GAA ATG GAT ATG.
      NOTE: A single PCR band of 250 bp indicates an R26Y homozygote, a single PCR band of 500 bp indicates a wild-type (WT), and two PCR bands (R26Y: 250 bp, WT: 500 bp) indicate an R26Y heterozygote (Figure 1A).
      For Trp53^L, perform PCR at 94 °C for 3 min, then at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min for 35 cycles, followed by 72 °C for 3 min, and maintaining at 14 °C. Use primers (i) p53F2-10 1F: CAC AAA AAC AGG TTA AAC CCA G; (ii) p53F2-10 1R: AGC ACA TAG GAG GCA GAG AC.
      NOTE: A single PCR band of 370 bp indicates a Trp53^L/L homozygote, a single PCR band of 288 bp indicates a Trp53^+/+ WT, and two PCR bands (WT: 288 bp, Trp53^L: 370 bp) indicate a Trp53^L/+ heterozygote (Figure 1B).

2. Preoperative preparation

1. Autoclave all surgical tools 1 day before the surgery.
2. Prepare 0.1% bromophenol blue in phosphate-buffered saline (PBS) and store it at 4 °C. Dilute the Ad-Cre that will be used for the intraductal injection in DMEM medium with 0.01 M CaCl₂ and bromophenol blue at a ratio of 1:10 (i.e., the injection mixture).
   NOTE: The Ad-Cre used here was obtained from the University of Iowa Viral Vector Core, with a stock viral titer of ~10¹⁰–10¹¹ pfu/mL.
3. Anesthetize the female mouse (F2 generation as described in step 1.2, age ranging from 3–4 weeks of age to adult) using an isoflurane chamber and apply eye ointment. During the procedure, anesthetize the mouse continuously by ensuring it inhales 1%–2.5% isoflurane in oxygen. Check the depth of anesthesia at least every 15 min by performing a toe pinch. Carefully monitor the mouse for any change in respiratory rate, adjusting the level of isoflurane accordingly, if needed.
4. Inject meloxicam as analgesia subcutaneously at a dose of 5 mg/kg, prior to the surgical procedure.
5. Expose the nipple surgical site by applying several drops of hair removal cream; remove excessive cream and loose hair using soft paper towels.
   NOTE: Perform this step in an area separate from where the surgery is to be performed. Shaving is not recommended in order to avoid damage to the nipples. Use caution to remove chemical depilatory agent in a timely fashion. Leaving the agent on for too long may result in chemical burn to the skin
6. Disinfect the surgical site with iodophors first, followed by 70% alcohol, and end with a final application of scrub iodophors. Do this in a circular motion from the center of the work area toward the periphery using a gauze sponge or cotton-tipped applicator. Repeat the cycle 3x–4x.

3. Intraductal injection

1. Use aseptic techniques throughout the surgical procedure.
2. Make an incision site on the skin at a length of ~1 cm between the two fourth inguinal MGs (Figure 2). Carefully separate the skin flap (with the MG) from the parietal peritoneum so as to visualize the mammary ductal tree.
3. Carefully hold the nipple with Watchmaker’s forceps and remove the exterior nipple without cutting any nearby skin, using a micro-dissection scissor.
4. Load ~3–5 µL of Ad-Cre injection mixture into a 25 µL Hamilton syringe with a 33 G metal hub needle affixed. Estimate the volume of the injection mixture in the syringe based on the blue dye included in the mixture.
   NOTE: Use a smaller volume (e.g., 3 µL) when injecting into MGs of 3–4 weeks old females and a larger volume (e.g., 10 µL) when injecting into MGs of lactating females.
5. Gently hold the edge of the skin flap with a fine curved tweezer and inject the Ad-Cre injection mixture slowly into the nipple, meanwhile monitoring the spreading of blue dye into the mammary ductal tree. Maintain the injection rate as low as possible to avoid damage to the ductal lumen.
   NOTE: Injected fluid (as illustrated by the included bromophenol blue dye) spreading throughout the entire ductal tree without leaking into the stromal compartment indicates a successful intraductal injection.
6. Gently withdraw the needle from the nipple to avoid any leakage of the injected fluid.
7. Examine the distal side (i.e., away from the nipple) of the MG or the surrounding area of the injected nipple. Note that swelling blue dye (i.e., dye diffusing into the nearby stroma) indicates a mammary fat pad injection rather than a successful intraductal injection.
8. Close the surgical wounds (from step 3.2) in the skin with wound clips.

4. Postoperative care

1. Remove the mouse from the anesthesia and place it on a heating pad inside a clean cage for recovery.
2. Administer meloxicam subcutaneously at 5 mg/kg again, 24 h after the surgery.
3. Monitor the general conditions of the animal and look for signs of infection at the incision site for 5 days.
4. Wound clips are removed ~7-10 days after surgery.

5. Monitoring the development of the mammary tumor

1. Monitor the injected mice twice weekly by palpation for any sign of mammary tumor development.
2. Once the tumor is palpable, monitor the mouse daily and measure the tumor size until it reaches the experimental endpoint, as determined by the size (e.g., reaching 10%–15% of the mouse’s body weight) or condition (e.g., ulcerated or necrotic) of the tumor, or by the general health condition of the mouse (e.g., comatose, moribund).
3. Euthanize the mouse by carbon dioxide asphyxiation, followed by a secondary method (e.g., cervical dislocation).
4. Isolate the mammary tumor tissues and analyze them by flow cytometry, immunofluorescence, or expression profiling (e.g., by RNA sequencing [RNAseq] or microarray), as described previously¹².
5. Perform flow cytometric analysis by gating for lineage-negative cells (Lin^-: negative for lineage markers CD45 [leukocyte marker], CD31 [endothelial cell marker], and TER119 [erythrocyte marker]), and analyze the cells in the tumor based on their expression of YFP, CD24, and CD29.

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