Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.
Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.
The intraductal delivery of therapeutic agents has been studied in rodent models and in early stage human trials3,4,5,6,11,12. A recent Phase I study demonstrated the safety and feasibility of intraductal carboplatin or intraductal pegylated liposomal doxorubicin in women awaiting mastectomy for the treatment of invasive cancer2.
Previous protocols for intraductal delivery have been developed for mouse and rat mammary glands6,7,8,9. For research purposes, intraductal tumor cell injections and the lentiviral vector delivery of oncogenes have also been performed in rodent models13,14,15,16. However, an ideal in vivo model of the intraductal delivery process should permit the development of novel classes of therapeutic compounds and facilitate preclinical assessment. Anatomical differences between rodents and humans have complicated the translation of these studies.
Unlike mice, in which each duct ends at a separate teat, the human breast consists of 5 to 9 independent ductal systems, each with a separate opening ending at the teat. Rabbit mammary glands harbor four independent ductal systems, each separately accessible through one of four orifices in a single teat. A rabbit model more closely matches the human anatomy and permits the study of intraductal drug delivery in a more relevant context.
Here, we use two techniques to assess intraductal delivery. The co-administration of a vital dye permits visualization through the skin and provides a simple and rapid confirmation of the method. For some applications, higher resolution mapping of the ducts may be preferred. We present here a protocol for ultrasound imaging of the ducts through the intraductal delivery of a non-targeted contrast reagent.
Procedures using animal subjects have been approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin.
1. Preoperative Preparation
2. Preparation of the Contrast Agent
3. Intraductal Delivery
4. Ultrasound Imaging
5. Postoperative Care
Here, we show that the intraductal delivery of contrast reagents to the mammary ducts of a rabbit can be achieved without trauma to the tissue (Figure 2). In rabbits, four separate ductal systems converge at one teat and thus may be accessed and imaged individually using this method. Individual ductal openings are easily visualized; note the arrowhead marking a second ductal opening adjacent to the cannulated duct in Figure 2B.
High-resolution ultrasound imaging with untargeted contrast reagent in linear imaging mode can provide a real-time readout of the intraductal delivery. Representative images show detection of the reagent up to 45 min post-delivery (Figure 3). This technique may also be useful for monitoring the kinetics of therapeutic delivery through the ducts.
The robustness of this injection method is highly dependent upon the operator. To master the injection technique, it is recommended to inject a solution of 0.2% Evans Blue dye and monitor the integrity of the gland. This provides the operator with an additional readout of success and also aids in the determination of the appropriate volumes to be injected into each gland. Simple visual assessment can be used to determine whether the dye reaches the entire ductal system (Figure 4) and whether any ducts are damaged during the delivery.
Figure 1: Schematic of Rabbit Mammary Glands. The two lower pairs of dots represent the teats of the inguinal glands. Please click here to view a larger version of this figure.
Figure 2: Preparation and Cannulation of the Inguinal Mammary Gland for Intraductal Delivery. (A) The teat of the right inguinal mammary gland is shown here immediately after the delivery of 0.2 mL of 0.9% sterile saline. Upon injection, the ductal openings are visualized more clearly. (B) A ductal opening in the same teat is then cannulated with a 25 G blunt-tip infusion needle. The arrow shows a ductal opening without a cannula. Please click here to view a larger version of this figure.
Figure 3. Non-targeted Contrast Reagent Visualized within the Mammary Duct by Ultrasound Imaging. (A) The contrast reagent is localized immediately after delivery and is visualized (B) 30 min post-delivery and (C) 45 min post-delivery. The persistence of the reagent inside the mammary duct allows visualization throughout the duration of this protocol. Please click here to view a larger version of this figure.
Figure 4: An Inguinal Mammary Gland Injected through the Teat with Evans Blue Saline Solution. (A) The external appearance after the intraductal injection of 0.2 mL of Evans Blue solution. (B) Upon opening the skin, the Evans Blue permits the visualization of the entire mammary ductal tree and confirms the intact ductal structure. (C) Whole-mount specimen of a region of inguinal mammary gland after fixation and staining with carmine alum. Please click here to view a larger version of this figure.
This method of intraductal delivery to the rabbit mammary gland may be used for ultrasound contrast reagents and many other aqueous solutions, including vital dyes and therapeutics. Previous studies have demonstrated the intraductal delivery of hormones17,18,19. In rodent models, the intraductal delivery of nucleic acids8, chemotherapeutics6,7, and nanoparticle carriers8,20 have been performed. The protocol described here could be adapted for these applications as well.
For some applications, the confirmation of the intraductal delivery by Evans Blue vital dye, as visualized through the skin, may be sufficient. However, visualization through the skin is diffuse, and individual ducts are not well demarcated. In endpoint studies, Evans Blue vital dye does provide a clear map of the entire ductal tree, but this requires the isolation of the mammary tissue. Therefore, contrast-enhanced ultrasound provides an alternative approach for visualizing the intraductal delivery to individual ducts in live animal studies. We note that Evans Blue dye maps the entire ductal tree, including the smallest-diameter terminal ducts, while contrast reagent and ultrasound maps only include the larger ducts. Another distinction is the possibility to monitor temporal dynamics in ultrasound, whereas Evans Blue provides only a single snapshot measurement.
As in the method for intraductal delivery to the rodent mammary duct9, the most significant challenge and limitation to this technique is likely to be the reliance on operator expertise. However, the larger size of the ductal openings in a rabbit model simplifies the procedure, eliminates the need for performing the technique with the aid of a stereomicroscope, and shortens the time required for new operators to develop proficiency. In our experience, the injection of 0.1-0.2 mL of saline to the side of the teat prior to the intraductal delivery is a critical step that enables the clear visualization of the ductal openings (step 3.2, above). Accurate positioning and lifting of the delivery site is also essential; this ensures that the solution flows into the duct (steps 3.5 and 3.6, above). We note that the co-administration of the contrast reagent will necessarily reduce the available volume for testing other reagents or therapeutics. However, intraductal delivery can also be performed without imaging or with simple inspection by Evans Blue to confirm the delivery.
The most common noninvasive lesion of the breast is ductal carcinoma in situ (DCIS), in which abnormal ductal epithelial cells proliferate inside the mammary duct but do not penetrate through the basement membrane to the adjacent tissue. With advances in mammographic imaging, the detection rates of DCIS have increased dramatically. In the United States, approximately 25% of newly diagnosed breast lesions are classified as DCIS, and by 2020, more than 1 million women will be living with DCIS in the United States alone22,23,24,25. However, many DCIS lesions remain dormant, and most estimates find that only 15-40%21,22,23,24,25 of DCIS lesions will ever progress to invasive cancer. However, there are currently no predictive biomarkers to aid in the identification of which tumors will become invasive.
As more women are diagnosed with this pre-cancerous lesion, serious questions regarding over-diagnosis and overtreatment have emerged. The treatment of premalignant disease is typically aggressive. Most patients with DCIS will undergo surgery (lumpectomy or mastectomy), and many also receive radiation25. Some patients with hormone receptor-positive DCIS will also receive 5 or more years of endocrine therapy, which has been shown to reduce recurrence. Side effects of this treatment may include stroke, blood clots, bone loss, and elevated risks of uterine and endometrial cancers. All of these options have serious systemic side effects and impact patient quality of life. There is a significant need for less invasive therapeutic strategies25.
The intraductal delivery of chemotherapeutic agents in both mouse models and in breast cancer patients has previously been shown to be effective, with no evidence of systemic toxicity or long-term histopathological changes3,4,5,6. The intraductal administration of therapeutics could one day offer new options for women diagnosed with DCIS that has not yet progressed to a locally invasive lesion. The potential for halting tumorigenesis while also preserving ductal structure makes this an attractive therapeutic strategy10. Importantly, the localized delivery approach ensures that the treatment reaches the relevant abnormal cells while potentially minimizing collateral damage to other tissues. While rabbit tumor models are not available, the normal mammary gland of rabbits may provide a relevant model to test the localized delivery, safety, transport, and kinetics of therapeutics uptake inside the mammary duct. These in vivo studies will enable the testing and validation of candidate diagnostics and therapeutics within a relevant tissue environment.
A similar approach enabled the intraductal administration of therapeutics to live mice and allowed for minimally invasive and localized drug delivery to the mammary ductal system8,9. However, the anatomy of the mouse mammary gland differs from the human breast in a number of important ways, including the tissue composition and the number of ducts that end at each teat. Here, we extend this technique to a larger animal model in which the mammary epithelial structure more closely represents the anatomy of the human breast12,14. This opens up the possibility for extended monitoring by imaging and for assaying the concomitant intraductal delivery of various reagents to the rabbit mammary ductal epithelium. Advances in localized delivery to an appropriate animal model, with ductal anatomy similar to the human gland, should accelerate the application of non-invasive, targeted therapeutic strategies in humans.
The authors have nothing to disclose.
The authors acknowledge support from a Translational Breast Cancer Research Grant (14-60-26-BROC to AB) from the Breast Cancer Research Foundation and the American Association for Cancer Research.
MicroMarker non-targeted contrast reagent | VisualSonics | VS-11694 | |
Luer Lock 1mL Syringes | BD | 309628 | |
Glycopyrrolate 0.2mg/mL | Wedgewood Compounding Pharmacy | GLYCOP-INJ013VC | 6 month shelf life, supply may be limited. |
Atropine Sulfate 0.5 mg/mL | Animal Health International | 15320764 | If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate. |
Ketamine HCL 100mg/mL | Animal Health International | 21250699 | http://www.animalhealthinternational.com/ |
Acepromazine 10mg/mL | Animal Health International | 17640541 | |
Xylazine 20mg/mL | Animal Health International | 20101547 | |
Yohimbine 0.2mg/mL | Animal Health International | 14588965 | |
Hair Removing Cream | Veet | Sensitive skin solution. Available through local retailers. | |
Blunt tip infusion needles | Sai Infusion Technology | B14-50 | http://www.sai-infusion.com/collections/blunt-needles |
Veterinary Pulse Oximeter | EdanUSA | VE-H100B | http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html |
Warm Water Pump | Gaymar | TP700 | |
Warm Water Blanket | Animal Health International | 21232696 | Maxi-Therm Lite Warming Pads |
Ultrasound system | VisualSonics | Vevo 2100 |