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 ...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laglambda-1:+A+Clinically+Relevant+Drug+Resistant+Human+Multiple+Myeloma+Tumor+Murine+Model+That+Enables+Rapid+Evaluation+of+Treatments+for+Multiple+Myeloma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+New+Model+of+Human+Multiple+Myeloma+Using+Nod/Scid/Gammac(Null)+(Nog)+Mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nod/Scid/Gamma(C)(Null)+Mouse:+An+Excellent+Recipient+Mouse+Model+for+Engraftment+of+Human+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Antitumor+Effects+of+the+Mtor+Inhibitor+Cci-779+against+Human+Multiple+Myeloma+Cells+in+a+Xenograft+Model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+Hypoxic+Bone+Marrow+Microenvironment+in+5t2mm+Murine+Myeloma+Tumor+Progression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-Inducible+Factor+(Hif)-1alpha+Suppression+in+Myeloma+Cells+Blocks+Tumoral+Growth+in+Vivo+Inhibiting+Angiogenesis+and+Bone+Destruction.">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. 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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 ...

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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6.6K vues.  Greater Los Angeles Veteran Administration Healthcare System. Cette méthode peut être utilisée pour répondre à des questions clés sur le rôle du micro-environnement de la moelle osseuse et la survie des tumeurs greffées du myélome. Le principal avantage de cette procédure est qu’elle peut être appliquée à des études antidrogue longitudinales et utilisée pour analyse de multiples voies biochimiques d’une manière non invasive. Le jour de l’expérience, lavez d’abord les 8226 cellules transfactées Luciferase trois fois dans le PBS ...

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