Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Summary

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Abstract

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal – particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

Introduction

In vitro studies of brain tumor cells are invaluable for dissecting molecular mechanisms driving growth, survival, migration, and invasion of cancer cells; cultured cell experiments can define signaling pathways, suggest potential therapeutic targets, and characterize cellular response to drug treatment. But in vitro systems are far too simplistic to predict organismal response to pharmaceuticals; they lack the physiological reactions, immune responses, cell microenvironment, and overall heterogeneity of living animal systems. Genetically engineered models can be invaluable, when available, but molecular differences exist between species and murine c....

Protocol

All described procedures were reviewed and approved by our institutional animal care and use committee.

1. Plan the Experiment

1. Choose cells to be injected. Cells from a variety of sources are candidates for injection: adherent cell culture lines, genetically modified clones, neurosphere cells, primary cultures, or disaggregated tumors. The type of model desired will define the most appropriate site of injection.
   1. Determine cell number for injection. The number of cells required to form tumors varies with cell line and must be determined empirically; tumor growth rate depends heavily on cell type, cell number and tissu....

Results

Reliable intracranial xenografts can be created with this described technique. Identifying the critical structures of the mouse skull (Figure 1) will allow for recognition of the bregma and guide the investigator to a precise and reproducible injection location. In these studies the U251 parental line, U251 cells transfected with luciferase (U251-Luc), or U87 immortalized human GBM tissue culture cells were suspended in 4 to 6 µl of SF-DMEM and injected 2.5 mm lateral (right), 1.5 mm anterior, and 3.5 mm.......

Discussion

Orthotopic mouse models of human brain cancer can be an excellent tool for assessing the effectiveness of clinical therapies, but care must be taken to optimize the placement of cells in brain tissue. Studies have shown that excessive aliquot volumes, suboptimal injection technique and hasty injection rates can lead to leakiness and the appearance of tumor cells in undesirable locations (ventricles, spinal cord, extradural regions, etc.) and high variation in tumor size² (personal observations). An an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment
Small Animal Stereotaxic Instrument with Digital Display Console	Kopf	Model 940
Mouse Gas Anesthesia Head Holder	Kopf	Model 923-B
Mouse Ear Bars	Kopf	Medel 922
Fiber Optic Illuminator	Fisher	12-562-36
UltraMicroPump III	WPI	UMP3
Micro4 microprocessor	WPI	UMC4
Variable speed hand-held rotary drill	Dremel	Model 300
Dental drill bit, 1.0 mm	Spoelting	514554
Adaptor for dental drill bit: 3/32 inch collet	Dremel	481
Heating pad			for mice
Isoflurane vaporizer system			for mice
Medical tubing and connectors			to connect isoflurane vaporizer with stereotaxic frame
Instruments
Precision 25 μl microsyringe	Hamilton	7636-01	Model 702, without needle
Microsyringe needles, 26s G	Hamilton	7804-04	RN, 25 mm point style 2
Fine-tipped scissors (straight, sharp/sharp)
Medium-sized standard scissors
Standard serrated forceps
Serrated hemostats (2)
Fine-tipped forceps
Supplies
Sutures 5-0 vicryl P-3 13 mm (Ethicon)	MWI	J463G
Surgical blades #10, stainless (Feather)	Fisher	296#10
Isoflurane (Fluriso)	VetOne	NDC 13985-528-60	Item #502017. Liquid inhalation anesthetic. federal law restricts this drug to use by or on the order of a licensed veterinarian.
Carprofen (Rimadyl Injectable 50 mg/ml)	Pfizer	NDC 61106-8507-01	dilute in saline
Ophthalmic ointment (artificial tears)	Rugby	NDC 0536-6550-91
Topical antibiotic (AK-Poly-Bac )	Akorn	NDC 17478-238-35
Povidone-iodine topical antiseptic, 10% (Betadine)	Betadine	NDC 67618-150-04
Hydrogen peroxide, 30%	Fisher	H325-100	for visualizing skull landmarks
Sterile saline	VetOne	NDC 13985-807-25	for diluting solutions, cleaning tissue
Bone wax	WPI	Item #501771
Sterile drapes	McKesson	25-517
Sterile surgical gloves	McKesson	(to fit)
Sterile gauze pads, 2 x 2	Fisherbrand	22028556
Sterile gauze pads, 4 x 4	Fisherbrand	22-415-469
Alcohol prep pads (medium)	PDI	B603
Sterile cotton-tipped applicators	Fisherbrand	23-400-114
Sterile 0.5 ml screw cap tube with caps for cells	USA Scientific	1405-4700	for cells
Individually wrapped sterile dispo pipettes	Fisher	BD 357575	for needle cleaning solutions
BD insulin syringes with needles	Fisher	329461	for analgesic
70% Ethanol			for cleaning
Sterile diH2O			for cleaning
Microfuge tubes for cleaning solutions			for needle cleaning solutions
Felt tip pen (dedicated)			for marking skull