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&#38;D Service (Merit Award I01CX001388) and VA Rehabilitation R&#38;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.

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.&#160;Preparation of Luciferase-expressing 8226 Cells (8226-LUC)
<ol>
	<li>Maintain the human MM cell line, 8226, in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C in a humidified atmosphere containing 95% air and 5% carbon dioxide (CO2).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Confirm the in vitro luciferase activity in 1 x 106 transfected cells/100 &#181;L of phosphate buffered saline (PBS) by adding the luciferase substrate, D-luciferin (150 &#181;g/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.</li>
</ol>
2.&#160;Orthotopic Xenograft Model
<ol>
	<li>Maintain NOG mice (4 - 6 weeks old, male or female) under sterile/pathogen-free conditions in an appropriate animal care facility.</li>
	<li>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 &#181;L of PBS) in a sterile test tube. Keep the cells on ice and challenge the mice as soon as possible (within 30 - 120 min).</li>
	<li>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.</li>
	<li>Inject the 8226-LUC cells into the mice through the tail vein (200 &#181;L/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.</li>
	<li>Return the animal to their home cage and monitor the animals until they have fully recovered.</li>
	<li>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).</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Give the animal an IP injection (200 &#181;L/injection) of &#8220;in vivo-grade&#8221; D-luciferin substrate (30 mg/mL diluted in sterile saline).</li>
	<li>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).
	<ol>
		<li>Measure the average radiance (photons/s/cm2/steradian) in selected regions of interest (ROIs) using imaging software (Figure 4).</li>
		<li>Return the animal to its home cage and monitor it until it has fully recovered (approximately 15 - 20 min).</li>
	</ol>
	</li>
</ol>
4. Treatment of the Mice with Targeted Therapy
<ol>
	<li>Measure the baseline BLI and randomize the animals into treatment groups (4 - 8 mice/group).</li>
	<li>Treat the mice with an IP injection (200 &#181;L/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.</li>
	<li>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.</li>
</ol>
5. PET/CT Analysis Using 18F-FDG to Measure Changes in Tumor Metabolism
<ol>
	<li>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.</li>
	<li>Prepare 18F-radiolabeled FDG PET probes (50 - 100 &#181;Ci/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.</li>
	<li>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.</li>
	<li>Inject the probe via the tail vein (100 &#181;L/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.</li>
	<li>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.</li>
	<li>Maintain the mice under anesthesia and at 37 &#176;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 &#176;C to avoid non-specific uptake of the probe.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

<ol><li>Raab, M. S., Podar, K., Breitkreutz, I., Richardson, P. G., Anderson, K. C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19541364">Multiple Myeloma.</a> The Lancet. 374 (9686), 324-339 (2009).</li>
<li>Galson, D. L., Silbermann, R., Roodman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+Multiple+Myeloma+Bone+Disease%5BTitle%5D)+AND+%22BoneKEy+Reports%22%5BJournal%5D)">Mechanisms of Multiple Myeloma Bone Disease.</a> BoneKEy Reports. 1, 135(2012).</li>
<li>Anderson, K. C., Carrasco, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+Myeloma%5BTitle%5D)+AND+%22Annual+Review+of+Pathology%22%5BJournal%5D)">Pathogenesis of Myeloma.</a> Annual Review of Pathology. 6, 249-274 (2011).</li>
<li>Reagan, M. R., Rosen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Navigating+the+Bone+Marrow+Niche%3A+Translational+Insights+and+Cancer-Driven+Dysfunction%5BTitle%5D)+AND+%22Nature+Reviews+Rheumatology%22%5BJournal%5D)">Navigating the Bone Marrow Niche: Translational Insights and Cancer-Driven Dysfunction.</a> Nature Reviews Rheumatology. 12 (3), 154-168 (2016).</li>
<li>Frost, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+Target+of+Rapamycin+Inhibitors+Induce+Tumor+Cell+Apoptosis+in+Vivo+Primarily+by+Inhibiting+Vegf+Expression+and+Angiogenesis%5BTitle%5D)+AND+%22Journal+of+Oncology%22%5BJournal%5D)">Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell Apoptosis in Vivo Primarily by Inhibiting Vegf Expression and Angiogenesis.</a> Journal of Oncology. 2013, 897025(2013).</li>
<li>Mysore, V. S., Szablowski, J., Dervan, P. B., Frost, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+DNA-Binding+Molecule+Targeting+the+Adaptive+Hypoxic+Response+in+Multiple+Myeloma+Has+Potent+Antitumor+Activity%5BTitle%5D)+AND+%22Molecular+Cancer+Research%22%5BJournal%5D)">A DNA-Binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma Has Potent Antitumor Activity.</a> Molecular Cancer Research. 14 (3), 253-266 (2016).</li>
<li>Podar, K., Chauhan, D., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+Marrow+Microenvironment+and+the+Identification+of+New+Targets+for+Myeloma+Therapy%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">Bone Marrow Microenvironment and the Identification of New Targets for Myeloma Therapy.</a> Leukemia. 23 (1), 10-24 (2009).</li>
<li>Campbell, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laglambda-1%3A+A+Clinically+Relevant+Drug+Resistant+Human+Multiple+Myeloma+Tumor+Murine+Model+That+Enables+Rapid+Evaluation+of+Treatments+for+Multiple+Myeloma%5BTitle%5D)+AND+%22International+Journal+of+Oncology%22%5BJournal%5D)">Laglambda-1: A Clinically Relevant Drug Resistant Human Multiple Myeloma Tumor Murine Model That Enables Rapid Evaluation of Treatments for Multiple Myeloma.</a> International Journal of Oncology. 28 (6), 1409-1417 (2006).</li>
<li>Miyakawa, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+a+New+Model+of+Human+Multiple+Myeloma+Using+Nod%2FScid%2FGammac%28Null%29+%28Nog%29+Mice%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Establishment of a New Model of Human Multiple Myeloma Using Nod/Scid/Gammac(Null) (Nog) Mice.</a> Biochemical and Biophysical Research Communications. 313 (2), 258-262 (2004).</li>
<li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nod%2FScid%2FGamma%28C%29%28Null%29+Mouse%3A+An+Excellent+Recipient+Mouse+Model+for+Engraftment+of+Human+Cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Nod/Scid/Gamma(C)(Null) Mouse: An Excellent Recipient Mouse Model for Engraftment of Human Cells.</a> Blood. 100 (9), 3175-3182 (2002).</li>
<li>Frost, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Vivo+Antitumor+Effects+of+the+Mtor+Inhibitor+Cci-779+against+Human+Multiple+Myeloma+Cells+in+a+Xenograft+Model%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">In Vivo Antitumor Effects of the Mtor Inhibitor Cci-779 against Human Multiple Myeloma Cells in a Xenograft Model.</a> Blood. 104 (13), 4181-4187 (2004).</li>
<li>Asosingh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+Hypoxic+Bone+Marrow+Microenvironment+in+5t2mm+Murine+Myeloma+Tumor+Progression%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Role of the Hypoxic Bone Marrow Microenvironment in 5t2mm Murine Myeloma Tumor Progression.</a> Haematologica. 90 (6), 810-817 (2005).</li>
<li>Storti, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia-Inducible+Factor+%28Hif%29-1alpha+Suppression+in+Myeloma+Cells+Blocks+Tumoral+Growth+in+Vivo+Inhibiting+Angiogenesis+and+Bone+Destruction%5BTitle%5D)+AND+%22Leukemia%22%5BJournal%5D)">Hypoxia-Inducible Factor (Hif)-1alpha Suppression in Myeloma Cells Blocks Tumoral Growth in Vivo Inhibiting Angiogenesis and Bone Destruction.</a> Leukemia. 27 (8), 1697-1706 (2013).</li>
<li>Fryer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+Novel+Mouse+Model+of+Multiple+Myeloma+and+Its+Use+in+Preclinical+Therapeutic+Assessment%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Characterization of a Novel Mouse Model of Multiple Myeloma and Its Use in Preclinical Therapeutic Assessment.</a> PLoS One. 8 (2), e57641(2013).</li>
<li>Gould, S. J., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Firefly+Luciferase+as+a+Tool+in+Molecular+and+Cell+Biology%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Firefly Luciferase as a Tool in Molecular and Cell Biology.</a> Analytical Biochemistry. 175 (1), 5-13 (1988).</li>
<li>Czernin, J., Phelps, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Positron+Emission+Tomography+Scanning%3A+Current+and+Future+Applications%5BTitle%5D)+AND+%22Annual+Review+Medicine%22%5BJournal%5D)">Positron Emission Tomography Scanning: Current and Future Applications.</a> Annual Review Medicine. 53, 89-112 (2002).</li>
<li>Pandey, M. K., Bhattacharyya, F., Belanger, A. P., Wang, S. Y., DeGrado, T. R. <a target="_blank" href="https://www.ahajournals.org/doi/abs/10.1161/circ.122.suppl_21.A12657">Pet Imaging of Fatty Acid Oxidation and Glucose Uptake in Heart and Skeletal Muscle of Rats: Effects of Cpt-1 Inhibition.</a> Circulation. 122 (21), (2010).</li>
<li>Wang, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+in+Vivo+Molecular+Imaging+Probe+%2818%29F-Annexin+B1+for+Apoptosis+Detection+by+Pet%2FCt%3A+Preparation+and+Preliminary+Evaluation%5BTitle%5D)+AND+%22Apoptosis%22%5BJournal%5D)">An in Vivo Molecular Imaging Probe (18)F-Annexin B1 for Apoptosis Detection by Pet/Ct: Preparation and Preliminary Evaluation.</a> Apoptosis. 18 (2), 238-247 (2013).</li>
</ol>

The author Kevin Francis is an employee of Perkin-Elmer.

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...

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 vivo5,6. 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 experiment7,8.
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 &#34;disseminated&#34; tumors that consistently and reproducibly engraft in the BM of NOD/SCID/IL-2&#947;(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.

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

multimodal bioluminescent and positronic-emission tomography/computational tomography imaging of multiple myeloma bone marrow xenografts in nog mice

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.

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...

Watch this Scientific Journal Video about Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice at JoVE.c

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

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.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

multimodal-bioluminescent-positronic-emission-tomographycomputational

Research

JoVE Journal

Cancer Research

6.8K Views.  Greater Los Angeles Veteran Administration Healthcare System. Deze methode kan worden gebruikt om belangrijke vragen te beantwoorden over de rol van de beenmergmicro-omgeving en de overleving van geëngrafeerde myeloomtumoren. Het belangrijkste voordeel van deze procedure is dat het kan worden toegepast op longitudinale anti-drug studies en gebruikt om meerdere biochemische trajecten te testen op een niet-invasieve manier. Op de dag van het experiment eerst wassen de 8226 Luciferase getransfacteerde cellen drie keer in ijskoude PBS op 200 RCF gedurende ...

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

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Medicine

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

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.

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 ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Bioengineering

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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

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