Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice
January 7th, 2019
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.
This method can be used to answer key questions about the role of the bone marrow micro-environment and the survival of engrafted myeloma tumors. The main advantage of this procedure is that it can be applied to longitudinal anti-drug studies and used to assay multiple biochemical pathways in a non-invasive manner. On the day of the experiment first wash the 8226 Luciferase transfacted cells three times in ice cold PBS at 200 RCF for five minutes per centrifigation and resuspend the pellet in fresh ice cold PBS at a five times ten to the sixth cells per 200 microliters of ice cold PBS per mouse.
Next confirm a lack of response to toe pinch in an anesthetized four to six week old NOG mouse and apply ophthalmic ointment to the animal's eyes. Load the cells into a one milliliter insulin syringe equipped with a 26 gauge needle. Inject the entire volume of cells into the tail vein of the animal, before returning the mouse to it's cage, monitoring until full recovery.
Ten to twenty days post challenge, inject each engrafted animal with 200 microliters of in vivo grade D-luciferin substrate in sterile saline intraperitonealy. Place the anesthetized animals in the supine position in a small animal imaging system within 5 to 10 minutes of the injection. Measure the average radiance in selected regions of interest within the corresponding imaging software.
For targeted therapy of the 8226 Luciferase tumors, After measuring the baseline bioluminescence in each animal as just demonstrated, randomize the animals into treatment groups and treat each mouse with a series of 200 microliter IP injection of Temsirolimus or a saline control. Measure the Luciferase activity two times per week and plot the changes in bioluminescence over time. To measure changes in the tumor metabolism, first remove the food from the home cage of the mice for 24 hours to avoid excess nonspecific radiolabeled Fludeoxyglucose uptake.
The next day dilute 500 to 100 microcuries of 18 Fluorine radiolabeled Fludeoxyglucose probe in sterile saline to a final volume of 100 microliters per mouse and record the time and activity of the probes with a dose calibrator. When the probes are ready place the first anesthetized animal on a heating pad with the head facing away and use a shielded one milliliter insulin syringe equipped with a 26 gauge needle to inject 100 microliters of the probe into the tail vein. Measure the residual radioactivity in the needle and syringe with the dose calibrator and note the activity and time.
Then measure the radiolabeled Fludeoxyglucose activity in selected regions of interest that correspond to the engrafted tumors in a small animal PET CT imaging system. A successful bone marrow tumor engraftment can be confirmed by bioluminescence imaging as demonstrated. Serial imaging of multiple animals can be used to visualize the distribution of the bone marrow engrafted multiple myeloma tumors.
Additionally optical imaging x-ray analysis shows a quick and non-invasive determination of the exact location and distribution of the multiple myeloma tumors within the mouse skeleton. The bioluminescence produced by these engrafted tumor cells can be serially and noninvasively measured to assess changes in tumor growth. Furthermore, the survival of mice treated with the mTOR inhibitor Temsirolimus can be monitored and positron emission and computed tomography analysis for tumor radiolabeled Fludeoxyglucose uptake can be used to demonstrate Temsirolimus mediated changes in glucose metabolism.
While performing this procedure it's important to inject the cells IEV. We've found that failure to inject the cells into the vein results in the formation of non-marrow engrafted tumor cells near the site of injection. Following this procedure, other methods like immunohistochemistry and micro-CT can be used to address questions about the tumors impact on bone architecture and the non-tumor cellular and molecular components of the marrow environment.
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Overview
This study utilizes bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to investigate the effects of mTOR inhibition on bone marrow-engrafted myeloma tumors in a xenograft model. This approach enables non-invasive, multimodal analyses of therapeutic impacts on myeloma tumors in vivo.
Key Study Components
Area of Science
- Oncology
- Immunology
- Biomedical Imaging
Background
- Bone marrow micro-environment plays a crucial role in tumor survival.
- Myeloma tumors can be engrafted in animal models for study.
- Non-invasive imaging techniques are essential for longitudinal studies.
- Understanding mTOR's role may lead to better therapeutic strategies.
Purpose of Study
- To assess the impact of mTOR inhibition on myeloma tumors.
- To explore the bone marrow's influence on tumor behavior.
- To develop a method for longitudinal anti-drug studies.
Methods Used
- Bioluminescent imaging of engrafted myeloma cells.
- X-ray imaging for anatomical visualization.
- Positron-emission tomography/computed tomography for metabolic assessment.
- Injection of Luciferase-transfected cells into NOG mice.
Main Results
- Inhibition of mTOR activity showed significant effects on tumor growth.
- Non-invasive imaging provided insights into tumor dynamics.
- Longitudinal studies revealed the role of the bone marrow micro-environment.
- Methodology allows for the assessment of multiple biochemical pathways.
Conclusions
- mTOR inhibition can effectively alter myeloma tumor behavior.
- Non-invasive imaging is a powerful tool for cancer research.
- Further studies are needed to explore therapeutic implications.
Frequently Asked Questions
Executive Industry Relevance
This multimodal imaging approach enables longitudinal, non-invasive assessment of tumor growth and metabolism in a physiologically relevant bone marrow microenvironment, addressing a critical gap in preclinical myeloma models. By combining bioluminescent and PET/CT readouts, the method supports mechanistic de-risking of therapeutic candidates targeting tumor-stroma interactions. It provides predictive confidence in target validation by linking mTOR inhibition to measurable changes in tumor bioluminescence and glucose metabolism over time.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by measuring changes in tumor bioluminescence as a direct readout of luciferase-expressing myeloma cell viability in vivo.
- Operational Value: Enables functional target validation through serial, non-invasive imaging that reduces reliance on endpoint histology.
- Predictive Value: Supports portfolio triage by correlating mTOR inhibition with sustained reductions in tumor signal, indicating target engagement and pathway modulation.
Screening & Assay Development
- Assay Readiness: Prepares validated xenograft models for downstream compound screening by establishing baseline bioluminescence and metabolic flux in engrafted tumors.
- Quantitative Outputs: Generates longitudinal bioluminescence and standardized uptake value (SUV) metrics from PET/CT, enabling dose-response and time-course analysis.
- Platform Reuse: Supports scalable evaluation of multiple biochemical pathways across therapy cycles without repeated animal sacrifice.
Translational & Preclinical Research
- Disease Relevance: Models the human multiple myeloma bone marrow niche, where tumor survival depends on stromal interactions not recapitulated in vitro.
- Translational Continuity: Links early target modulation (via BLI) to downstream metabolic effects (via FDG-PET), supporting biomarker-aligned go/no-go decisions.
- Risk-Adjusted Advancement: Enables longitudinal monitoring of therapeutic impact, reducing late-stage failure by confirming mechanism of action in a clinically relevant microenvironment.
Pipeline & Workflow Integration
The method integrates into the discovery continuum from target validation through lead optimization, providing orthogonal validation of pharmacological activity in a disease-relevant system.
- Discovery Biology: Supports hypothesis testing by quantifying tumor burden and metabolic activity in response to pathway inhibitors, clarifying target dependence in the bone marrow niche.
- Screening: Delivers assay-ready models with reproducible engraftment and detectable signal, enabling high-fidelity compound evaluation across multiple time points.
- Analytics: Provides dual-modality readouts—bioluminescence for tumor volume and FDG-PET for metabolic flux—allowing teams to disaggregate cytostatic from cytotoxic effects.
- Translational Research: Connects early pharmacodynamic changes to preclinical efficacy endpoints, supporting continuity from mechanism to outcome.
- Enterprise Reuse: Establishes a reusable imaging platform for evaluating stroma-dependent targets across oncology portfolios, maximizing ROI on model development.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by correlating target inhibition with concurrent changes in tumor growth and glucose metabolism.
- Operational Value: Standardizes longitudinal data collection through reproducible imaging protocols, minimizing inter-animal variability.
- Strategic Value: Improves go/no-go decisions by providing early, quantitative evidence of target-mediated tumor modulation, reducing investment in ineffective candidates.
- Portfolio Impact: Enables risk-adjusted prioritization of compounds that demonstrate sustained target engagement in a microenvironmentally accurate model.
Implementation Considerations
- Requires expertise in xenograft modeling, bioluminescent imaging, and small-animal PET/CT operation and analysis.
- Dependent on access to imaging systems capable of bioluminescence and PET/CT, along with radiolabeled FDG and D-luciferin handling infrastructure.
- Necessitates cross-team standardization between in vivo pharmacology, imaging, and data analysis groups to ensure consistent region-of-interest selection and quantification.
- Involves adaptation considerations when extending to other tumor models, as engraftment efficiency and signal stability may vary by cell line and microenvironment.
- Practical limitations include signal attenuation from overlying tissues and the requirement for fasting prior to FDG injection to minimize nonspecific uptake, as noted in the protocol.
