Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Summary

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Abstract

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Introduction

Brain metastasis is a prevalent malignancy associated with a very poor prognosis^1,2. The standard of care for brain metastasis patients is multimodal, consisting of neurosurgery, whole brain radiotherapy and/or stereotactic radiosurgery depending on the patients' general health status, extracranial disease burden, and the number and location of tumors in the brain^3,4. Patients with up to three intracranial lesions are eligible for surgical resection or stereotactic radiosurgery, while whole-brain radiation therapy is recommended for patients with m....

Protocol

All studies were conducted within the guidelines of the Animal Ethics Committee of The University of Queensland (UQCCR/186/19), and the Australian Code for the Care and Use of Animals for Science Purpose.

1. Preparation of cancer cells for injection

NOTE: In this study, the human breast cancer cell line, BT-474 (BT474), was used. BT474 was cultured in complete growth medium comprising RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% in.......

Discussion

Brain metastasis is a complex process of cancer cells spreading from their primary site to the brain. Different animal models are available that mirror certain stages of this multi-step process and there are physiological and practical considerations to designing preclinical metastasis studies^41,42. Most published studies investigating the use of nanomedicine for brain metastasis treatment have used intracardiac^43,

Materials

Name	Company	Catalog Number	Comments
100µm cell strainer	Corning	CLS431752
30G Microlance needle	BD	23748
31G Ultra-Fine II insulin syringe	BD	326103
Angled forceps	Proscitech	T67A-SS	Fine pointed, angled without serrations, 18mm tip, length 128 mm
Animal heat mat
Antibiotic and antimycotic	ThermoFisher Scientific	15240062
Autoclave bags
BT-474 (HTB-20) breast cancer cell line	ATCC	HTB-20
Buprenorphine (TEMGESIC)
Countess cell counter	ThermoFisher Scientific	C10227
Diet-76A	ClearH2O	72-07-5022
Dissection microscope
Ear puncher
Electric clippers
Fine angled forceps	Proscitech	DEF11063-07	Angled 45°, Tip smooth, Tip width: 0.4 mm, Tip dimension: 0.4 x 0.3 mm, length 9cm
Fine tubing for cannula, Tubing OD (in) 1/32, Tubing ID (in) 1/100in	Cole Parmer	EW-06419-00
Foetal bovine serum	ThermoFisher Scientific	26140079
Hank's Balanced Salt Solution without calcium and magnesium	ThermoFisher Scientific	14170120
Hydrogel	ClearH2O	70-01-5022
Isoflurane
Kimwipes Low lint disposable wipers	Kimberly Clark- Kimwipes	Z188964
Mashed mouse chow
Meloxicam (METACAM)
Nose cone			Fashioned out of a microfuge tube
PAA ocular lubricant (Carbomer 2mg/g)	Bausch and lomb
Povidone-iodine solution	Betadine	2505692
PPE (glove, mask, gown, hairnet)
Retractors	Kent Scientific	SURGI-5001
RPMI 1640 Media	ThermoFisher Scientific	11875093
Silk suture 13mm 5-0, P3, 45cm	Ethicon	JJ-640G
Sterile normal saline	ThermoFisher Scientific	TM4469
Sticky tape
Surgical board			A chopping board wrapped with autoclavable bag.
Surgical scissors	Proscitech	T104	Tip Dimensions (LxD): 38x7mm, Length 115mm
Suture forcep/ Curved Brophy forceps	Proscitech	T113C	Curved, Rounded narrow 2 mm tip, with serrations, length 165 mm
Suture needle holder (Olsen Hegar needle holder)	Proscitech	TC1322-180	length 190 mm, ratchet clamp
Syringe driver with foot pedal/ UMP3 Ultra micro pump	World Precision Instruments	UMP3-3
T75 tissue culture flask	ThermoFisher Scientific	156499
Thread
Trigene II surface disinfectant	Ceva
Trypan Blue and Cell Counting Chamber Slides	ThermoFisher Scientific	C10228
TrypLE Express dissociating medium	ThermoFisher Scientific	12605010