Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

Summary

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

Abstract

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

Introduction

Animal models play a key role in cancer research. When a hypothesis is proven in vitro, it should be tested in vivo to evaluate its clinical relevance. Cancer progression and metastasis are often better captured by animal models as compared to in vitro models, and it is essential to test a new drug in an animal model as a preclinical study for drug development^1,2. However, the technical details of animal experiments are often not well described in published articles, making it challenging to reproduce the model successfully. Indeed, the authors who established these orthotopic inoculation and mastectomy models....

Protocol

Approval from the Roswell Park Comprehensive Cancer Center Institutional Animal Care and Use Committee was obtained for all experiments.

NOTE: Nine to twelve weeks-old female BALB/c mice are obtained. 4T1-luc2 cells, a mouse mammary adenocarcinoma cell line derived from BALB/c mice that has been engineered to express luciferase, are used. These cells are cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS).

1. Preparation of Instruments

1. Thaw a frozen gelatinous protein mixture (e.g., Matrigel) on ice in a tissue culture hood.
2. ....

Results

The purpose of the orthotopic model is to mimic human cancer progression (i.e., the growth of the primary tumor followed by lymph node metastasis and then distant lung metastasis)¹⁵. After cancer cell inoculation, the bioluminescence is quantified regularly (two to three times/week) (Figure 1A). The bioluminescence in the lungs is deeper and smaller than the primary lesion. The bioluminescence mainly reflects the primary tumor.......

Discussion

For the last decade, we have been establishing multiple murine cancer models, including breast cancer models^{3,7,13,16,20,21}. Previously, we demonstrated that breast cancer cell orthotopic inoculation into the mammary gland tissue under direct vision produced a larger tumor with less size variability compared to injecting cell.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Micro Dissection Scissors	Roboz	RS-5983	For cancer cell inoculation and masstectomy
Adson Forceps	Roboz	RS-5233	For cancer cell inoculation and masstectomy
Needle Holder	Roboz	RS-7830	For cancer cell inoculation and masstectomy
Mayo	Roboz	RS-6873	For ex vivo
5-0 silk sutures	Look	774B	For cancer cell inoculation and masstectomy
Dry sterilant (Germinator 500)	Braintree Scientific	GER 5287-120V	For cancer cell inoculation and masstectomy
Clipper	Wahl	9908-717	For cancer cell inoculation and masstectomy
Matrigel	Corning	354234	For cancer cell inoculation
D-Luciferin, potassium salt	GOLD-Bio	LUCK-1K	For bioluminescence quantification
Roswell Park Memorial Insitute 1640	Gibco	11875093	For cell culture
Fetal Bovine Serub	Gibco	10437028	For cell culture
Trypsin-EDTA (0.25%)	Gibco	25200056	For cell culture