Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis
July 29th, 2017
Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.
We have developed a new murine model of de novo mammary cancer metastasis that closely mimics the course of human breast cancer, providing a preclinical platform for evaluating the biology, underlying metastatic progression of disease, and the efficacy of therapies delivered in the adjuvant setting. Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University. Our murine model is established by harvesting enzymatically dissociated primary tumors from transgenic 100-day old mouse mammary tumor virus polyoma middle T or MMTV-PyMT mice.
One times 10 to the sixth tumor cells are orthotopically injected into syngeneic recipient female mice and primary tumors develop over a 38 to 60-day period. Mice bearing tumors 170 to 450 millimeters cubed are enrolled for surgical resection of the primary tumor. Analysis at 115 days postsurgery resulted in mice that have histologically detectable metastatic foci in the lungs with 45%penetrance.
This video depicts surgical resection of the primary mammary tumor, a crucial component of the preclinical murine model. Mice are administered 2%isoflurane and surgery can commence once the foot pinch reflex is absent. Spray the skin overlying the primary tumor with 70%ethanol.
Betadine is then applied to the surgical area beginning on the skin overlying the tumor followed by the area surrounding the primary tumor using a sterile cotton swab. Using blunt-tip scissors, an initial incision is made in the skin medial caudal to the primary tumor. The incision is extended in a superior direction medial to the primary tumor.
Identify and cauterize potential vessels feeding the tumor from the overlying skin. If bleeding occurs, further cauterization should be performed. When making a lateral skin incision inferior to the primary tumor, make sure to avoid cutting the underlying mammary gland as vessels may be present.
The skin incision is then extended circumferentially around the primary tumor. Thin adhesions in the mammary gland medial to the tumor are then carefully dissected separating the tumor from the abdominal wall. Blood vessels may be present within these adhesions.
Cauterize any vessels identified within the adhesions prior to cutting. Arrows indicate the fourth and fifth right mammary glands, shown above and below the tumor. The fifth mammary gland is then cauterized and excised as it may contain small vessels that are difficult to visualize.
After the tumor has been separated from the abdominal wall, larger vessels can be identified. The vessel running through the fourth mammary gland is shown by the top arrow. The underlying femoral artery is shown by the middle arrow.
And the large vessel feeding the tumor between the fourth and fifth mammary gland is indicated by the bottom arrow. Cauterization along the large vessel running between the fourth and fifth mammary gland is then performed, making sure to avoid cauterization of the underlying femoral artery on the leg. After cauterization of the large vessel is complete, cut through the vessel up to the fourth mammary gland.
Locate and cauterize the second large vessel running through the fourth mammary gland. Cut through the vessel without cutting through the entire mammary gland so that if bleeding occurs, the vessel can be easily identified and further cauterized. The remainder of the fourth mammary gland is then excised.
The overlying skin is then closed with wound clips using an AutoClip wound clip applier. Make sure the overlying skin is sufficiently separated from the abdominal wall to avoid clipping the underlying muscle. Bring the lateral and medial edges of the wound together, lifting the skin in an anterior direction to apply the wound clip.
Make sure to avoid clipping the urogenital tract or underlying muscle of the right leg. Mobilization of the right leg by removing tape may allow for easier application of wound clips. Wound clips should be removed 10 days following surgery.
The surgical resection of the primary tumor displayed in this video allows for disseminated cancer cells to develop in the metastatic foci in the lung 115 days postsurgery where a representative of each in the image of a metastatic foci is shown. Given that a large percentage of women diagnosed with breast cancer undergo surgical removal of the tumor and can succumb to metastatic disease postsurgery, we established a preclinical murine model that mimics the course of patient disease and treatment. By recording the methodology for surgical removal of the primary mammary tumor, this key component of our murine model of de novo mammary cancer metastasis can be easily reproduced in order to assess metastatic disease biology post tumor resection and adjuvant therapy efficacy.
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Overview
This article presents a detailed protocol for establishing a murine model of de novo mammary cancer metastasis. By surgically resecting primary mammary tumors in transgenic mice, researchers can study metastatic progression and evaluate adjuvant therapies in a setting that closely mimics human breast cancer. The model enables investigation of late-stage metastasis and therapeutic efficacy post-tumor resection.
Key Study Components
Area of Science
- Cancer biology
- Metastasis research
- Preclinical animal models
Background
- Traditional transgenic mouse models of mammary adenocarcinoma are limited by primary tumor burden, which often dictates study endpoints.
- This limitation hampers research into late-stage metastasis and the evaluation of therapies targeting metastatic disease after primary tumor removal.
- Many women with breast cancer receive adjuvant therapy following surgical resection, highlighting the need for relevant preclinical models.
- Existing models do not adequately mimic the clinical course of metastatic progression post-surgery.
Purpose of Study
- To develop a murine model that allows for the study of de novo mammary cancer metastasis after surgical removal of the primary tumor.
- To provide a platform for evaluating the biology of metastatic progression in the absence of primary tumor burden.
- To facilitate preclinical testing of adjuvant therapies targeting metastatic foci.
Methods Used
- Harvesting enzymatically dissociated primary tumors from MMTV-PyMT transgenic mice.
- Orthotopic injection of 1 x 106 tumor cells into syngeneic female mice.
- Allowing primary tumors to develop over 38–60 days, followed by surgical resection when tumors reach 170–450 mm3.
- Detailed surgical procedure including anesthesia, aseptic preparation, careful dissection, cauterization of vessels, and wound closure with clips.
- Post-surgical monitoring and removal of wound clips after 10 days.
Main Results
- At 115 days post-surgery, 45% of mice developed histologically detectable metastatic foci in the lungs.
- The model provides a long latency period for studying metastatic progression in the absence of primary tumor.
- Representative images confirm the presence of metastatic foci in the lungs.
- The protocol is reproducible and suitable for evaluating the efficacy of adjuvant therapies.
Conclusions
- This murine model closely mimics the clinical course of human breast cancer, particularly the development of metastasis after primary tumor resection.
- It enables functional studies of metastatic progression and therapeutic intervention in a clinically relevant context.
- The detailed surgical methodology ensures reproducibility for future research on metastatic disease and adjuvant therapy efficacy.
Frequently Asked Questions
Executive Industry Relevance
Late-stage metastasis remains a critical challenge in oncology drug discovery, with traditional murine models limited by early study endpoints due to primary tumor burden. This surgically enabled preclinical model allows for the isolation and longitudinal study of de novo metastatic progression post-tumor resection, directly aligning with clinical scenarios of adjuvant therapy in breast cancer. The model supports predictive confidence in evaluating anti-metastatic agents and informs risk-adjusted portfolio decisions for late-stage therapeutic candidates.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of molecular drivers of metastatic progression in the absence of primary tumor confounders.
- Supports functional validation of targets implicated in metastatic survival and colonization.
- Facilitates mechanistic de-risking by modeling clinically relevant metastatic latency.
Screening & Assay Development
- Provides a reproducible in vivo system for evaluating anti-metastatic compound efficacy post-surgery.
- Standardizes metastatic endpoint assessment through histological detection of lung foci.
- Enables quantitative comparison of therapeutic interventions in a controlled metastatic context.
Translational & Preclinical Research
- Aligns preclinical testing with patient treatment paradigms involving adjuvant therapy after tumor resection.
- Supports translational biomarker discovery by enabling longitudinal sampling post-metastasis initiation.
- Improves predictive value for clinical progression and therapeutic response in metastatic disease.
Pipeline & Workflow Integration
This model bridges early discovery and preclinical validation by enabling hypothesis-driven studies of metastatic biology and therapeutic efficacy after primary tumor removal.
- Discovery Biology: Isolates metastatic mechanisms for target validation and pathway analysis post-resection.
- Screening: Establishes a platform for reproducible, quantitative assessment of anti-metastatic agents.
- Analytics: Delivers histological and temporal readouts for comparing metastatic burden across cohorts.
- Translational Research: Models clinical adjuvant therapy scenarios, supporting biomarker and efficacy studies.
- Enterprise Reuse: Offers a standardized, scalable model for cross-program metastatic research.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in anti-metastatic target validation and therapeutic assessment.
- Operational Value: Enhances reproducibility and standardization of metastatic endpoint evaluation.
- Strategic Value: Informs go/no-go decisions for late-stage oncology assets targeting metastasis.
- Portfolio Impact: Enables risk-adjusted prioritization of candidates with anti-metastatic potential.
Implementation Considerations
- Requires surgical expertise and adherence to animal care protocols for reproducibility.
- Necessitates access to histological and imaging infrastructure for metastatic endpoint analysis.
- Demands cross-team standardization of surgical and post-operative procedures.
- Adaptation to other cancer models may require protocol optimization for tumor type and metastatic site.
- Latency and penetrance rates should be considered when planning cohort sizes and study timelines.
