Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Summary

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.

Abstract

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women¹. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Introduction

Women in North America have a ~12% lifetime risk of developing breast cancer²; a majority of these individuals will have primary tumors removed via surgery, and depending on cancer subtype, will then receive targeted, endocrine, chemo- and/or radiation therapy in the adjuvant setting³. Examples include, women diagnosed with hormone receptor-positive cancers receiving anti-estrogen therapies and women with HER2-positive tumors receiving HER2-targeted therapies with radiation/chemotherapy, whereas no targeted therapies are yet available for triple negative tumors³. Despite advances in radiation, che....

Protocol

Animals used in the following protocol are covered by Oregon Health & Science University's Institutional Animal Care and Use Committee (IACUC), which is designed to be compliant with the Animal Welfare Act regulations and Public Health Service (PHS) Policy.

Maintenance of sterile conditions: Sterilized instruments should be used and between mice, should be wiped clean with sterile gauze, rinsed in PBS followed by sterilization with disinfectant 70% ethanol for at least 15 minutes. A surgical cap, facemask, gown, and gloves should be worn for survival surgeries. Pre-operative preparation of the animal for survival surgeries is includ....

Results

Greater than 75% of recipient mice receiving 1 x 10⁶ cells from primary mammary tumors derived from MMTV-PyMT mice, develop single mammary adenocarcinomas ranging in size from 172 to 450 mm³ within 38-60 days (data not shown). Mice eligible for randomization are then enrolled into study groups following surgical resection of primary tumors as shown (Figure 1C). Primary tumor regrowth was identified in less than 2% of mice that underwent .......

Discussion

Modifications and troubleshooting:

When blunt dissecting tumor away from the abdominal wall, the tumor may remain adherent to the abdominal wall. This was observed in <5% of mice injected with tumor (data not shown). For mice with tumors adherent to the abdominal wall, the mouse should be euthanized as resection is difficult without primary tumor regrowth.

Limitations of model/technique:

Whereas other.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isofluorane	Piramal Healthcare	N/A	Prescription order
Collagenase A	Roche	11088793001
DNase I	Roche	10104159001
DMEM	ThermoFisher	12634010
25 mL Pyrex bottle	Sigma-Aldrich	CLS139525
Fetal Bovine Serum	Atlanta Bio	S11150
0.7 µm nylon strainer	Corning	352350
50 mL conical tube	VWR	89039-658
Dimethyl sulfoxide	Sigma-Aldrich	D2650
Growth factor-reduced Matrigel	BD	354230	Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma
Poly(vinylpyrrolidone)–Iodine complex	Sigma-Aldrich	PVP1
29 gauge 0.3 mL insulin syringe	BD	324702
Small Vessel Cauterizer Kit	FST	18000-00
Wound clips	Texas Scientific	205016
AutoClip wound clip applier	BD	427630
AutoClip wound clip remover	BD	427637
Bromodeoxyuridine	Roche	10280879
Heparinized capillary tubes	Fisher	22362566
Microtainer tubes with dipotassium EDTA	BD	365974
20 mL syringe	BD	309661
DPBS	Thermo-Fisher	14190-250
OCT-freezing medium	VWR	25608930
Formalin, Buffered, 10% (Phosphate Buffer)	Fisher	SF100-4
23g needle	Fisher	14-826-6B
FVB/n mouse	Jackson Laboratories	001800