Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Summary

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Abstract

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(¹⁸F)fluoro-D-glucose (¹⁸F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Introduction

MM is an incurable disease made up of malignant plasma B-cells that infiltrate the BM and cause bone destruction, anemia, renal impairment, and infection. MM makes up 10% - 15% of all hematological malignancies¹ and is the most frequent cancer to involve the skeleton². The development of MM stems from the oncogenic transformation of long-lived plasma cells that are established in the germinal centers of lymphoid tissues before eventually homing to the BM³. The BM is characterized by highly heterogeneous niches; including diverse and critical cellular components, regions of low pO₂ (hypoxia), extensive vascularization, complex extracellular matrices, and cytokine and growth factor networks, all of which contribute to MM tumorgenesis⁴. Thus, the development of a disseminated MM xenograft model characterized by tumors that are strictly engrafted in the BM would be a very powerful and clinically relevant tool to study MM pathology in vivo^5,6. However, numerous technical hurdles can limit the effectiveness of most xenograft models, making them costly and difficult to apply. This includes problems associated with consistent and reproducible tumor engraftment within the BM niche, a prolonged time to tumor development, and limitations in the ability to directly observe and measure changes of tumor growth/survival without having to sacrifice mice during the course of the experiment^7,8.

This protocol uses a modified xenograft model that was initially developed by Miyakawa et al.⁹, in which an intravenous (IV) challenge with myeloma cells results in "disseminated" tumors that consistently and reproducibly engraft in the BM of NOD/SCID/IL-2γ(null) (NOG) mice¹⁰. The in situ visualization of these tumors is achieved by the stable transfection of the 8226 human MM cell line with a LUC allele and serially measuring the changes in the BLI produced by these engrafted tumor cells⁶. Importantly, this model can be expanded to utilize various other LUC-expressing human MM cell lines (e.g., U266 and OPM2) with a similar propensity to specifically engraft in the skeleton of NOG mice. The identification of the tumors by bioluminescent imaging of the mice is followed by measuring the uptake of radiopharmaceutical probes (such as ¹⁸F-FDG) by PET/CT. Together, this allows for additional characterization of critical biochemical pathways (i.e., alterations in metabolism, changes in hypoxia, and the induction of apoptosis) within the tumor/BM microenvironment. The major strengths of this model can be highlighted by the availability of a wide range of radiolabeled, bioluminescent and fluorescent probes and markers that can be used to study MM progression and pathology in vivo.

Protocol

All animal procedures described below were approved by the Institutional Animal Care and Use Committee (IACUC) of the Greater Los Angeles VA Healthcare system and were performed under sterile and pathogen-free conditions.

1. Preparation of Luciferase-expressing 8226 Cells (8226-LUC)

1. Maintain the human MM cell line, 8226, in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified atmosphere containing 95% air and 5% carbon dioxide (CO₂).
2. Generate stable LUC-expressing reporter 8226 cells by transfecting 1 x 10⁶ 8226 cells with a luciferase reporter vector followed by a selection with hygromycin (100 - 350 mg/mL) as previously described⁶.
3. Maintain the cells under standard sterile culturing conditions for 2 - 3 weeks, changing the medium every other day, until the establishment of a stably transfected 8226 cell line has been generated.
4. Confirm the in vitro luciferase activity in 1 x 10⁶ transfected cells/100 µL of phosphate buffered saline (PBS) by adding the luciferase substrate, D-luciferin (150 µg/mL working solution in prewarmed culture medium), and measuring the bioluminescence of the cells in a test tube or 96-well plate using a luminometer⁶.

2. Orthotopic Xenograft Model

1. Maintain NOG mice (4 - 6 weeks old, male or female) under sterile/pathogen-free conditions in an appropriate animal care facility.
2. Prepare the 8226 LUC-transfected cells for IV injection into the NOG mice (~5 x 10⁶ cells/mouse) on the day of the experiment. Wash the cells 3x in ice-cold PBS, count them, and resuspend them in ice-cold PBS (minus antibiotics and FBS) at a final concentration of 5 x 10⁶ cells/200 µL of PBS) in a sterile test tube. Keep the cells on ice and challenge the mice as soon as possible (within 30 - 120 min).
3. Anesthetize the mouse (with isoflurane, 1% - 3% in air at 0.5 - 1 L/min) and place the animal in a supine position on a heating pad with the head facing away. Check the level of anesthetization by pinching the toe. Apply ophthalmic ointment to the eyes.
4. Inject the 8226-LUC cells into the mice through the tail vein (200 µL/injection) using a 1 mL insulin syringe and a 26-G needle.
   Note: A heat lamp can be used to warm the tail, thereby dilating the vein and increasing the efficacy of injections.
5. Return the animal to their home cage and monitor the animals until they have fully recovered.
6. Confirm the engraftment of 8226-LUC cells in the mouse skeleton by measuring the BLI in anesthetized mice (described below and shown in Figure 1A).
   Note: BM exudates can also be stained for human CD45 expression using flow cytometry (Figure 1B) or by the immunohistochemistry of harvested bone (Figure 1C).

3. Measurement of BLI In Vivo

Note: Typically, measurable BLI from engrafted tumors in mice can be observed first between 10 - 20 days postchallenge, but this may need to be experimentally determined.

1. Anesthetize the mouse (with isoflurane, 1% - 3% in air at 0.5 - 1 L/min) and check the level of anesthetization by pinching the toe. Apply ophthalmic ointment to its eyes.
2. Give the animal an IP injection (200 µL/injection) of “in vivo-grade” D-luciferin substrate (30 mg/mL diluted in sterile saline).
3. Measure the BLI within 5 - 10 min, following the injection of the luciferin substrate (although the BLI activity can be observable in the mice for up to 45 - 60 min), by placing the anesthetized animal in a supine position in a small-animal imaging system (Figure 2). Select luminescent and X-ray analysis and acquire images (Figure 3).
   1. Measure the average radiance (photons/s/cm²/steradian) in selected regions of interest (ROIs) using imaging software (Figure 4).
   2. Return the animal to its home cage and monitor it until it has fully recovered (approximately 15 - 20 min).

4. Treatment of the Mice with Targeted Therapy

1. Measure the baseline BLI and randomize the animals into treatment groups (4 - 8 mice/group).
2. Treat the mice with an IP injection (200 µL/injection) of temsirolimus (0.2 - 40 mg/kg mouse) using a regimen of five daily IP injections followed by 2 days of rest and an additional five daily injections¹¹.
3. Measure the luciferase activity (photons/s/cm²/steradian) 2x per week as described in section 3 and plot the change of the BLI over time. Changes in tumor BLI can be presented as serial images of the mice (Figure 5B).
   Note: It is recommended that daily monitoring of the animals be performed until they present the following symptoms (typically within 45 - 60 days of the initial challenge): weight loss (loss of more than 10% relative to the weight prior to implantation) and/or hind limb paralysis, at which time they should be euthanized using a CO₂ chamber followed by cervical dislocation.

5. PET/CT Analysis Using ¹⁸F-FDG to Measure Changes in Tumor Metabolism

1. Fast the animals in their home cage by removing their food for 24 h prior to the experiment to avoid excess non-specific uptake of ¹⁸F-FDG.
2. Prepare ¹⁸F-radiolabeled FDG PET probes (50 - 100 µCi/injection in sterile saline) on the day of the experiment. Record the time and activity of the ¹⁸F-FDG probe with a dose calibrator.
   Note: Because ¹⁸F has a relatively short half-life (~2 h), a careful preparation and planning for the use of these probes must be established.
3. Anesthetize the animal (with isoflurane, 1% - 3% in air at 0.5 - 1 L/min) and place it in a supine position on a heating pad with the head facing away. Check the level of anesthetization by pinching the toe. Apply ophthalmic ointment to its eyes.
4. Inject the probe via the tail vein (100 µL/injection) using a shielded 1 mL insulin syringe and a 26-G needle.
   Note: A heat lamp can be used to warm the tail, thereby dilating the vein and increasing the efficacy of injections.
5. Measure the residual radioactivity in the needle/syringe in a dose calibrator and note the activity and time.
   Note: The standardization of mouse incubation, dosage, and timing is essential to ensure data reproducibility and comparability.
6. Maintain the mice under anesthesia and at 37 °C using a heating pad during the whole period of probe uptake (~45 - 90 min).
   Note: It is important that the mice remain quiescent and at 37 °C to avoid non-specific uptake of the probe.
7. Stagger the injections of the ¹⁸F-FDG probe to occur at approximately 20- to 30-min intervals to ensure similar labeling times in the mice.
   Note: Proper workflow and timing of the probe injections will result in optimal and reproducible imaging conditions.
8. Measure the ¹⁸F-FDG activity in a PET/CT small-animal imaging system in selected regions of interest that correspond to the engrafted tumors (Figure 6).
   Note: Initially, a 10-min static PET exposure and a 2-min CT exposure are recommended, but the exact exposure times may need to be experimentally determined.
9. Return the animals to their home cages and monitor them until fully recovered. House the mice in a dedicated return room for 24 h while the probe decays to background levels.
   Note: Because of the short half-life of ¹⁸F, multiple measurements using a fresh probe can be made as frequently as 2x per week.

Results

In initial pilot studies, IV injections of 8226-LUC cells into NOD/SCID mice did not develop BM-engrafted MM tumors, although squamous MM tumors were easily formed (100% success rate). In contrast, IV challenges with 8226 cells into NOG mice generated (within 15 - 25 days) tumors in the skeleton (and only rarely formed tumors in non-skeletal tissue, such as the liver or spleen). Since tumors in the skeleton could not be visually confirmed by physically examining the animals, other methods...

Discussion

Despite a variety of preclinical xenograft models of MM^6,9,11,12,13, the ability to study the tumor/BM interactions within the BM microenvironment remains difficult¹⁴. The techniques described here allow for the rapid and reproducible engraftment of 8226-LUC tumors cells in the skeleton of NOG mice.

The criti...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
8226 human myeloma cell line	ATCC	CCL-155
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ Mice (NOG)	Jackson Labs	5557
VivoGlo Luciferin substrate	Promega	P1041
Hypoxyprobe-1Kit	HPL	HP1-100
PE-CD45 (clone H130)	BD Biosciences	555483	Used for flow cytometry to identify human CD45+ tumor cells in BM exudate
rabbit anti-human CD45 (clone D3F8Q)	Cell Signaling Technology	70527	Primary antibody used for Immunohistochemistry of excised bone
Goat Anti-rabbit IgG (HRP conjugated)	ABCAM	ab205718	Seconday antibody used for Immunohistochemistry of excised bone
Dual-Luciferase Reporter Assay System	Promega	E1910
pGL4.5 Luciferase Reporter Vector	Promega	E1310
IVIS Lumina XRMS In Vivo Imaging System	Perkin Elmer
Sofie G8 PET/CT Imaging System	Perkin Elmer