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.
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Method Article
Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice
In This Article
Summary
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 (hypo....
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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)
- 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).
- Generate stable LUC-expressing reporter 8226 cells by transfecting 1 x 106 8226 cells wit....
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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.......
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Discussion
Despite a variety of preclinical xenograft models of MM6,9,11,12,13, 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.......
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Disclosures
The author Kevin Francis is an employee of Perkin-Elmer.
Acknowledgements
This work was supported by a VA MERIT grant1I01BX001532 from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service (BLRDS) to P.F., and E.C. acknowledges support from the VA Clinical Science R&D Service (Merit Award I01CX001388) and VA Rehabilitation R&D Service (Merit Award I01RX002604). Further support came from a UCLA Faculty Seed Grant to J.K. These contents do not necessarily represent the views of the US Department of Veterans Affairs or the US Government.
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Materials
| 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 |
References
- Raab, M. S., Podar, K., Breitkreutz, I., Richardson, P. G., Anderson, K. C. Multiple Myeloma. The Lancet. 374 (9686), 324-339 (2009).
- Galson, D. L., Silbermann, R., Roodman, G. D. Mechanisms of Multiple Myeloma Bone Disease. BoneKEy Reports. 1, 135(2012).
- Anderson, K. C., Carrasco, R. D.
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