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 a drug, procedure, or treatment to be effective prior to testing in humans. One of the earliest laboratory steps in preclinical studies is identifying potential molecular targets for drug treatment by using cancer cells implanted in a host organism, defined as human xenograft models. Within this context, intracranial brain tumor xenograft models using patient-derived xenografts (PDXs) have been widely used to study brain tumor biology, progression, evolution, and therapeutic response, and more recently for biomarkers development, drug screening, and personalized medicine^2,3,4.

One of the most affordable and non-invasive in vivo imaging methods to monitor intracranial xenografts is bioluminescence imaging (BLI)^5,6,7,8. However, some BLI limitations include substrate administration and availability, enzyme stability, and light quenching and scattering during imaging acquisition⁹. Here we report the infrared FMT as an alternative imaging method to monitor preclinical glioblastoma models. In this method, signal acquisition and quantification of intracranially implanted PDXs, expressing a near-infrared fluorescent protein iRFP720^10,11 (henceforth termed as FP720) or turboFP635 (henceforth termed as FP635), is performed with a FMT imaging system. Using the FMT technology, the orthotopic tumors can be monitored in vivo before, during, or after treatment, in a non-invasive, substrate-free, and quantitative manner for preclinical observations.

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 (FBS) in a 15-cm dish; or culture 2.0 x 10⁶ human glioblastoma patient-derived (GBM-PDX) spheres with DMEM/F12 1:1 medium with B27 supplement plus human recombinant epidermal growth factor (EGF; 20 ng/mL) and FGF (10 ng/mL) in a T75 flask.
3. Maintain all cells at 37 °C, 5% CO₂, and 100% relative humidity.
4. Dissociate the cells.
   1. For the glioma cells: remove the supernatant from the plates and adding 3-5 mL of trypsin 1X to the glioma cells.
   2. For the GBM spheres: collect the cells by pipetting the cells into a 15-mL tube.
      1. Spin down the GBM spheres at 200 x g for 5 min using a tabletop centrifuge at room temperature.
      2. Remove the supernatant and add 3-5 mL of cell detachment solution.
   3. Incubate all cells at 37 °C for 10-15 min.
5. Carefully dissociate the cells by pipetting up and down several times (ensure that spheres and glioma cells are completed dissociated) and spin down at 200 x g for 5 min.
6. Remove the supernatant and resuspend the cells with 5 mL of medium by pipetting up and down several times. Determine the cell viability by trypan blue exclusion.
7. Plate 1.0 x 10⁶ of target cells in a 10-cm dish with 5 mL of medium by pipetting.
8. Transduce cells with lentivirus expressing fluorescent proteins at multiplicity of infection (MOI) 5 as is described by Tiscornia et al.¹² and incubate at 37 °C.
9. After 24 h, remove the medium from the transduced cells, add 5 mL of fresh medium, and incubate for additional 48 h at 37 °C.
10. Dissociate the infected cells into a single-cell suspension as described in steps 1.4-1.6. Spin down the cells at 200 x g for 5 min and resuspend in 500 µL of sorting solution (phosphate-buffered saline (PBS) with 1% FBS).
11. Pipette the cell suspension into FACS tubes. Co-stain the cells with 1 µL/mL of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride to exclude dead cells. Negative controls are required to set up the flow cytometer gates (non-transduced cells and non-transduced cells/ DAPI stained).
12. Sort the fluorescent-positive/DAPI-negative cells into sorting solution, as described by Basu et al.¹³, and collect the sorted cells into a 15-mL conical tube.
13. Spin down the sorted cells at 200 x g for 5 min, remove the supernatant, and resuspend in 5 mL of culture medium. Seed the sorted cells in a 10-cm dish by pipetting. Incubate at 37 °C for at least 48-72 h.
14. Expand the sorted cells by dissociating the cells following steps 1.4-1.6 and plating into multiple dishes for in vivo experiments.
    NOTE: For more details about cell sorting preparation and purification, see Basu et al.¹³

2. Intracranial Injection of iRFP-tagged Glioblastoma Cells into Immunodeficient Mice

NOTE: Before starting the surgery, make sure that the surgical room and tools are clean for the procedure. Use immunodeficient athymic nude (Foxn1^nu) male or female mice, between 4-5 weeks old and 17-19 g for intracranial injections. Animals should be housed for at least 3 days after arrival and before surgery.

1. Cell preparation
   1. Harvest and dissociate fluorescent positive-glioma cells into a single-cell suspension (steps 1.4-1.6) and resuspend 0.5 or 1.0 x 10⁶ cells in 2-5 µL of PBS per injection per animal. Pipette the cell suspension into a 1.5-mL microcentrifuge tube and place on ice.
2. Anesthesia preparation and administration
   1. Prepare 200 µL of saline solution containing ketamine (100 mg/kg) and xylazine (10 mg/kg) per 20 grams of mouse body weight. Weigh the animals and calculate the appropriate dose of anesthesia.
   2. Provide an intraperitoneal injection of anesthesia and apply ophthalmic ointment to the eyes to prevent dehydration. For more details about this step see Kathleen et al.¹⁴ Place the animal on a pre-warmed water thermal pad at 37 °C.
   3. Check that the animals are anesthetized by monitoring the toe pinch response (pedal reflex), usually within 5-10 min.
3. Intracranial injection
   1. Load a 5 µL Hamilton syringe (blunt tip needle) with cell suspension and mount in the probe holder.
   2. Place the mouse in a small animal stereotactic frame and fix the head using the ear bars and the incisor adapter following the described details by Cetin et al.¹⁵
   3. Disinfect the head with 70% ethanol and betadine solution. Make sure that the surgical site is complete sterile. Perform a middle incision with a small scalpel and separate the skin and connective tissues.
   4. Place the Hamilton syringe on the bregma point using the micromanipulator.
   5. Move the probe holder 1.0 mm anteroposterior and 2.0 mm lateral from the bregma point. Mark this position with a pencil.
   6. At this location, carefully make a hole in the skull with a micromotor hand-held drill by applying slight pressure downward until the blood vessels become visible. Do not disrupt the arachnoid mater and brain parenchyma.
   7. Introduce the needle into the burr hole and advance 3.0 mm below the pial surface.
   8. Set up the volume (2-5 µL) and flow rate (1 µL/min) of injection using a motorized stereotaxic injector or manually perform the injection.
   9. After the injection, remove the needle gradually by ascending 1.0 mm every 1 min and clean the injection site with 70% ethanol.
   10. Close the skin wound with surgical staples or by making surgical sutures (often referred to as stitches) and see Kathleen et al.¹⁴ for more details.
4. Animal recovery
   1. Transfer the animal to a water thermal pad (37 °C) and monitor the body temperature, respiration rate, and heart rate, until full recovery. Administer analgesia per standard protocols.
      NOTE: For more information about the stereotactic procedure, surgery, and coordinates, see other reports^5,14,15.

3. FMT Imaging

NOTE: According to the experimental aim, the iRFP-tagged glioblastoma cells can be monitored in vivo before, during, or after treatment. For imaging purpose, anesthetize animals using an isoflurane induction chamber, maintain in an imaging cassette during the scanning, and image in the docking station of the FMT imager.

1. Remove the animal from the cage and place in the isoflurane induction chamber. Close the chamber and turn on oxygen flow and vaporizer to 3-5%.
2. Monitor the animal until it is completely anesthetized, usually within 1-2 min. Unconscious animals should not respond to external stimuli.
3. Remove the anesthetized animal from the chamber and put the animal in the imaging cassette by placing the head adaptor first, followed by placing the animal facing down. Make sure that the head is located in the center of the cassette.
4. Place the top plate of the cassette on top and tighten the adjustment knobs until 17 mm.
5. Once that the animal is secure in the imaging cassette, proceed to imaging by inserting the cassette into the internal docking station of the FMT imager, where the isoflurane is pumped to keep the animal anesthetized.
6. Run the imager and analyzer software by double-clicking the software icon.
7. In the "Experimental tab" window, select the "Database" and "Study group".
8. Go to the "Scan tab" window and select the subject to image by clicking "select subject". Also, select the laser channel in the "Laser channel" panel. For the FP635-labeled cells, select "laser channel 635 nm", and for FP720-labeled cells, select "laser channel 680 nm".
9. Acquire an image by clicking the "Capture" option in the "Scan tab" window. Then, adjust the scan field by clicking and dragging the scan field in the captured image to a determined number of source locations, usually within 20-25 sources for the ipsilateral side.
10. Check the "Add to reconstruction queue" option and hit "Scan" in the "Scan tab" window.
11. Wait until the scanning is completed and then remove the imaging cassette from the docking station.
12. Remove the top plate of the cassette and place the animal back into the cage.
13. Repeat the steps 3.1-3.12 for the rest of the animals.

4. Image Analysis

1. From the imager and analyzer software, select the "Analysis tab" window.
2. Load the dataset and the subject to analyze by clicking the "+" button in the "Dataset selection" panel of the "Analysis tab" window.
3. Once the image has been loaded in the "Analysis tab" window, perform the region of interest (ROI) analysis by selecting the ellipsoid icon, located in the upper left corner of the "Analysis tab" window.
4. Adjust the threshold to zero in the "statistic data" panel by right clicking the "threshold" column in the "Analysis tab" window.
5. The total value in the "Statistic data" panel represents the signal from the fluorescent-tagged glioblastoma cells implanted in the mouse brain.
6. Repeat steps 4.2-4.4 for the rest of the animals. Load or remove a new subject by clicking the "+" or "−" button, respectively, in the "Dataset selection" panel of the "Analysis tab" window.
   NOTE: For more information about the FMT imaging and software operation, see^20.

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

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.