Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Summary

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

Abstract

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs' metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Introduction

Breast cancer is the leading cause of female mortality worldwide. With its progressively increasing incidence, breast cancer has become a serious challenge to public health¹. Murine cancer models are good bridges between preclinical and clinical studies, and a good mimic murine disease model will increase the accuracy of research on disease and medicine.

Primary tumor growth starts the progress of malignant disease, while metastasis and complications are the main causes of death and poor life qualities in most cancer patients. Several murine models are used to mimic the pathology of human breast cancer

Protocol

Animal experiments were conducted in accordance with the Provision and General Recommendation of Chinese Experimental Animal Administration Legislation and were approved by the Institution of Animal Care and Use Committee of Capital Medical University (Ref no. AEEI-2014-052). Female Balb/c mice aged 6 to 8 weeks were used.

1. Preparation of the Cells and Animals

1. One day before the operation, shave the hair around the fourth nipples to expose the operating area.
   1. Use one hand to hold the mouse tightly at the back to make sure the mouse will not move freely, turn up the mouse to expose its belly to the operator, and the....

Results

After a successful injection, one white transparent flat sphere with the nipple as the out-surface round center can be observed (Figure 1). Primary tumor growth can be measured by tumor volume and living tumor cell bioluminescence (Figure 2). Both the tumor volume and the total flux increased during the experiment before resection. At an early stage, metastasis cannot be found because no secondary tumor has happened or the strong.......

Discussion

The cells used in this study are 4T1-luc2, murine triple-negative breast cancer cells with luciferase labeling, which are a useful tool for investing antitumor and antimetastatic effects of drugs due to their highly invasive nature^2,19. Luciferases, stable to the next cell generation, are used to indicate the living tumor cells, both at the mammary gland and at other distant organs²⁰. In some cases, hypoxia and nutrient deficiency within t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
anesthesia machine	Midmark Corporation, Dayton, OH, USA	Matrx VMS	anesthesia
In-Vivo Imaging System	PerkinElmer	IVIS Spectrum	used for bioluminescence detecion
isoflurane	Hebei yipin chemical reagents  company	O21400	anesthesia
1 ml syringe	Becton,Dickinson and  Company	A257	cell injection
digital caliper	Shang Hai Shen Han Measuring Tools Co., Ltd.	S-H	volume measurement
tramadol	Mundipharma company	-	pain killer
D-luciferin	Gold Biotechnology Inc.	LUCK-1G	used for bioluminescence detecion
The primary antibody against cluster of differentiation (CD) 31	Abcam	ab28364	used for MVD detection
hematoxylin and eosin staining kit	Beijing Zhong Shan Jinqiao Biotechnology Co., Ltd.	ZLI-9615	histology
hair removal cream	Veet	-	hair removal cream
Carbomer Eye Gel	Dr.GerhardMannChem-Pharm.FabrikGmbH	-	ophthalmic ointment
sewing needle	Shanghai Pudong Jinhuan Medical Products Co., Ltd.	17U0302J	suture