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^2,3,4. Xenograft models are widely used for cancer study to understand the pathological characters and to screen drugs for safety and efficacy^5,6,7. Genetically engineered mice (GEM) are generated to mimic human breast cancer by targeting certain oncogenes or tumor suppressor genes⁸. GEM have a relatively simple and uniformed background to understand the role of genes in cancerous progress; however, the artificial environment and background are limited to investigate the metastasis pathology and related therapies⁹. Human cancer cells, although with human pathological features, can only be implanted in immune-deficient mice, and the insufficiency of the tumor-host immune interaction may lead to biased results¹⁰.

Where the solid tumor initiates has a direct influence on the biological and pathological characters of the disease^11,12,13. Since cancerous progress is the complicated outcome of interactions among tumor cells, stromal cells, immunology cells, inflammatory cells, growth factors, and proteases, primary tumors implanted in situ will provide better insight and mimic the cancerous process more accurately than tumors induced by chemical agents or a subcutaneous injection of tumor cells. Chemical agents used to induce tumors may be harmful to researchers and environment and are even forbidden in some countries. Because of the absence of a mammary fat pad environment, the pathological progress of a subcutaneous injection may differ with that in real breast cancer patients, whose cancer originates and irritates from the mammary fat pad. The disadvantages of subcutaneous injections encourage the use of orthotopic models to study tumor growth. In earlier research, highly metastatic MDA-MB-231 tumors, developed after seven orthotopic transplantations, indicated the importance of the injection location¹⁴. Recently, the orthotopic implantation of breast cancer cells into the mammary fat pad with surgery was reported^15,16. With the mammary pad environment, the tumor growth and the migration into distant organs cover the entire process of breast cancer at pathologically relevant sites, which makes this model a miniature of the progress of human diseases. However, after the surgery, the skin automatically attempts to heal itself, which may bring the potential risk of interfering with the normal breast cancer origination and bias the results.

We have compared some breast cancer models and established a minimally invasive orthotopic model to investigate the potential effect of drugs on breast cancer progression^17,18. In this study, a video protocol on how to orthotopically inject breast cancer cells into the mammary fat pad in a simple, less invasive way is presented. This orthotopic injection method without surgery is advantageous in many ways. First, the operation is simple and rapid, about 1 minute per mouse. Second, with primary tumor foci starting at the right pathological sites, it covers the whole tumorigenic process of breast cancer progression from the tumor growth to other organs metastasis, which provides a good experimental animal model for studying the interaction of tumor cells and tumor microenvironment. Besides, it can be a valuable model to estimate the treatment effects at all stages of breast cancer. The goal of this method is to provide an animal model to maximumly mimic human breast cancer progression.

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 then use another hand to shave the fur around the fourth nipples with an electronic shaver.
   2. Put a thin layer of hair removal cream around the nipples for 30 - 60 s, clean away the cream with distilled water, and then dry the mouse with soft paper.
2. On the day of the operation, collect murine breast cancer cells (4T1-luc2), count the cell number, and adjust the cell density to 2 x 10⁵ cells/mL in phosphate-buffered saline (PBS) in sterile microcentrifuge tubes and keep them on ice. For each mouse, 1 x 10⁴ cells/50 µL in a 1-mL syringe is prepared.

2. Orthotopic Injection

1. Anesthesia of mice
   1. Induce anesthesia. Put the mice in a closed transparent container and turn on 2% isoflurane gas to anesthetize the mice for about 5 min, till they all lie down and fall asleep.
   2. Maintain anesthesia. Transfer the mice from the container to individual anesthesia masks, put some vet ointment on their eyes to avoid dryness, keep the mice supinely with their nipples exposed to the operator, and let them continue to inhale isoflurane gas for a few minutes until the injection has been performed.
   3. Confirm the success of anesthesia by the lack of a toe pinch withdrawal reflex.
   4. Apply ophthalmic ointment to the eyes of the mice to prevent eye dryness.
      Note: According to the container size and the number of individual anesthesia masks available, a group of mice can be anesthetized together and injected one by one under individual anesthesia.
2. Performance of the injection
   1. Use one hand to tent up the skin around the fourth nipple with tweezers, to set up an uplifted “tunnel” for the syringe to follow. Use the other hand to hold the syringe with the needle turned upwards to enter the skin subcutaneously at 5 - 10 mm from nipple; then, follow the “tunnel” until the needle tip comes close to the nipple, carefully move the needle tip up into the mammary fat pad till the needle eye comes under the nipple tip, discard the tweezers, and inject the cells slowly.
   2. Turn around the needle a little and retract the syringe slowly; use a cotton swab to press the needle wound for 10 - 20 s to make sure there is no fluid seepage.
   3. Observe the fourth nipple to confirm a successful injection: one white transparent flat sphere with the nipple as the center can be observed (Figure 1).
3. Keep the mice anesthetized for another minute to avoid letting the mice recover and move too quickly, which will cause the injection wound to open and the tumor cells to leak out.

3. Analysis of the Tumor Growth and Metastasis

Note: Primary tumors are identified as transparent or blister-like bumps after injections, and as spherical or ellipsoid solid lumps with the nipple a few days later. Tumor growth is evaluated by tumor volume and tumor bioluminescence.

1. To establish the tumor volume, hold the mouse with one hand. Expose its belly to the operator and use the other hand to caliper the tumor length (L) and width (W). Calculate the tumor volume (V) as follows.
   V = (L x W²)/2
2. To capture the tumor bioluminescence, inject 150 mg/kg D-luciferin at the back of the mouse’s neck, 10 min before imaging. Anesthetize the mice with 2% isoflurane gas for 5 min and transfer them to individual anesthesia masks to let them inhale isoflurane gas for 5 min longer. Following the manufacturer’s instructions, capture bioluminescence images of the primary tumor and metastasis, with auto exposure time, medium binning, and f/stop at 1.

4. Harvesting of the Primary Tumor and Metastasis Organs for Analysis

Note: When achieving the purpose of this experiment, or reaching the humane endpoint, the primary tumor can be harvested for further study. In this study, tumors are removed at 4 to 5 weeks after the injection, when the tumor volume reaches about 800 mm³. The lungs are removed at week 8.

1. Harvesting of the primary tumor
   1. Perform the surgery in a sterile hood to maintain a sterile atmosphere. Anesthetize the animals by isoflurane. Confirm the success of anesthesia by the lack of toe pinch withdrawal reflexes. Apply ophthalmic ointment to the eyes of the mice to prevent them from drying out.
   2. Dissociate the tumor from the skin with scissors, dropwise painkiller (1 mg/mL tramadol, 1 drop of about 50 µL per mouse) to relieve postoperative pain.
   3. Stitch the skin with a surgical suture, cut off excess suture, drape off the incision.
   4. Cut the primary tumor in half with a scalpel, put 1/2 of the tumor into 4% paraformaldehyde for further hematoxylin and eosin (H&E) staining and immunohistochemistry, and immediately freeze the other 1/2 with liquid nitrogen for western blotting or an RNA isolation study later.
   5. After suturing, warm the mice with a lamp till they recover, keep the mice in single cages until fully recovered. Continuously administer 10 mg/kg tramadol with an oral gavage for 3 d after the surgery.
2. Harvesting of metastasis organs
   1. Euthanize the mice with an overdose of pentobarbital, open their chests with scissors, gently take out the lungs, and wash them with PBS. Lung metastasis foci can be identified as white transparent bumps which are morphologically different and distinguishable from normal pink lung tissue.
   2. Put the lungs into 4% paraformaldehyde to verify the metastasis by H&E staining.

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