Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Summary

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Abstract

Metastasis, the spread and growth of malignant cells at secondary sites within a patient's body, accounts for > 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Introduction

The metastasis of cancer to the central nervous system (CNS) is a devastating disease, and can involve either the brain parenchyma or the leptomeninges ("brain metastasis" refers to both in this article). It is the predominant intracranial malignancy, outnumbering primary gliomas by > 10:1^1,2. Lung cancer, breast cancer, and melanoma are the top three major neoplastic diseases that produce high incidences of brain metastasis^3,4. In recent years, impressive advances in novel cancer therapies have dramatically prolonged survival and improved ....

Protocol

Ethics statement: All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Texas MD Anderson Cancer Center.

1. Prepare Cancer Cells for Injection

1. Seed the cancer cells one or two days before injection. Use DMEM/F12 media supplemented with 10% FBS, unless a specialty medium is indicated in literature for a particular cell line.
2. On the day of surgical operation, harvest cells when they reach 70 - 80% confluence by first washing with serum-free media once before trypsinization (0.25%) for 1 - 2 min at 37 °C. Add into cells 10% FBS-containing DMEM/F12 media to quench the 0.25% trypsi....

Results

There are two points at which the quality of injection can be evaluated. The first chance is by the operator's observation of changing blood vessel colors during injection. Leakage from poor injections can be easily observed under the dissecting microscope. The steady and secure placement of the mouse body (Figure 1A) and its carotid artery (Figure 1B) on the supporting cotton ball are critical factors for smooth and successful injection into the carotid .......

Discussion

The most critical steps for successful carotid arterial injection of cancer cells are: 1) deep anesthesia of mouse in preparation for surgical operation; 2) steady placement of carotid artery on top of the cotton support; 3) tight ligation of carotid artery after successful injection.

Deep anesthesia of ~ 30 min is usually necessary for steady surgical performance under the dissecting microscope. We use commercially ready-to-use ketamine/xylazine cocktail for mouse anesthesia. To avoid potenti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine hydrochloride/xylazine hydrochloride solution	Sigma-Aldrich	K113
Routine Stereomicroscope Leica M50	Leica	M50	The microscope is modular and highly configurable to fit particular space requirements.
Surgical disposable scapel	Integra Miltex	4-410	to make skin incisions
Tissue Forceps - 1 x 2 Teeth	Fine Science Tools	11021-12	to bluntly dissect mucles
Dumont #5 - Mirror Finish Forceps	Fine Science Tools	11252-23	to separate and prepare the carotid artery for injection
Spring Scissors	Fine Science Tools	15025-10	to cut sutures
EZ Clip Kit	Stoelting	59020	for wound cloure
BD Insulin Syringe	Becton Dickinson	328438	for cell injection
IVIS Spectrum In Vivo Imaging System	PerkinElmer	124262