Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Summary

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Abstract

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Introduction

Cancer is one of the leading causes of illness-related deaths in humans in the industrialized world. With an extremely high death toll, new treatments are urgently required. Glioblastoma multiforme (GBM) is an extremely lethal type of brain cancer, composed of heterogeneous populations of brain tumor, stromal, and immune cells. According to the Central Brain tumor registry of the USA, the incidence of primary malignant and non-malignant brain tumors is approximately 22 cases per 100,000. Approximately 11,000 new cases are expected to be diagnosed in the USA in 2017¹.

Preclinical studies investigate the likelihood of ....

Protocol

The use of experimental research animals and infectious agents, such as lentivirus to transduce the cancer cells, require prior approval by the institutional animal care program and by the institutional biosafety committee. This protocol follows the animal care guidelines of the University of California San Diego (UCSD).

1. Labeling of Glioblastoma Cells with FP635 or FP720 Construct

1. Produce and purify lentivirus according to the protocol described by Tiscornia et al.¹²
2. Culture approximately 2.0 x 10⁶ cells from a human glioma cell line with DMEM plus 10% fetal bovine serum (....

Results

Glioblastoma cells U87EGFRvIII (U87 cells over-expressing the EGF receptor variant III) were cultured according to the step 1.2. Lentivirus was produced and purified according to step 1.1. The viral concentration was determined by p24 ELISA analysis. Cells were transduced with lentivirus carrying infrared fluorescent proteins according to step 1.8. The plasmid encoding FP720^10,11 was kindly provided by Dr. V.V. Verkhusha and the F.......

Discussion

Tumor xenografts have been extensively used in cancer research and a number of well-established imaging techniques has been developed: BLI; magnetic resonance imaging (MRI); positron emission tomography (PET), computed tomography (CT); FMT. Each of these approaches comes with pros and cons, but ultimately complement each other with the type of information provided. One of the most commonly used in vivo imaging technology is BLI^5,6,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/High Glucose	HyClone/GE	SH30022.1
DMEM/F12 1:1	Gibco	11320-082
FBS	HyClone/GE	SH30071.03
Accutase	Innovative cell technologies	AT-104
Trypsin	HyClone/GE	SH30236.01
B27 supplement	Gibco	17504044
human recombinant EGF	Stemcell Technologies	2633
human recombinant FGF	Stemcell Technologies	2634
DPBS	Corning	21-031-00
FACS tubes	Falcon	352235
DAPI	ThermoFisher Scientific	62248
Blasticidin	ThermoFisher Scientific	A1113903
p24 ELISA	Clontech	632200
Xylazine	Akorn	NDC 59399-110-20
Ketamine	Zoetis	NADA 043-403	Controlled substance
Ointment	Dechron	NDC 17033-211-38
Absorbable suture	CpMedical	VQ392
5 ul syringe	Hamilton	26200-U	Catalog number as sold by Sigma-Aldrich
Cell Sorter	Sony	SH8007
Mouse stereotaxic frame	Stoelting	51730
Motorized stereotaxic injector	Stoelting	53311
Micromotor hand-held drill	Foredom	K1070
Mouse warming pad	Ken Scientific Corporation	TP-22G
Fluorescence Tomography System	PerkinElmer	FMT 2500 XL
TrueQuant Imaging Software	Perkin Elmer	7005319
Ultra-centrifuge Optima L-80 XP	Beckman Coulter	392049
Tissue Culture 100mm Dishes	Olympus Plastics	25-202
Tissue Culture 150mm Dishes	Olympus Plastics	25-203
Tissue Culture Flasks T75	Corning	430720U
50 mL conical tubes	Corning	430290
15 mL conical tubes	Olympus Plastics	28-101
Centrifuge Avanti J-20	Beckman Coulter	J320XP-IM-5
Tube, Polypropylene, Thinwall, 5.0 mL	Beckman Coulter	326819
Tube, Thinwall, Polypropylene, 38.5 mL, 25 x 89 mm	Beckman Coulter	326823
Athymic nude mice	Charles River Laboratories	Strain Code  490 (Homozygous)	Prior approval by the Institutional Animal Care Program and by the Institutional Biosafety Committee required.