Name: a xenograft mouse model to assess efficacy of therapeutic agents for human acute leukemia
Uploaded: 2020-01-14T11:19:58
Description: Preclinical evaluation of therapeutic agents using an appropriate animal model is a critical step and a requirement for selecting drugs worth testing in ...

This study was funded by the Kenneth F Noel Memorial Fund (D.F.C.), Delbert J. McQuaide Cancer Research Fund (A.S.), Austin R. Orwan Memorial Research Fund (A.S.), The Penn State Cancer Institute (PSCI) and the National Institutes of Health (NIH) under the National Cancer Institute (T.P.L.) (P01CA171983). This work was supported by Penn State Clinical and Translational Sciences KL2 award (KL2 TR002015) to CG. The authors thank the staff of the Penn State Cancer Institute, Department of Comparative Medicine, Bioluminescence Imaging, Flow Cytometry, and Four Diamonds Developmental Therapeutic Preclinical Core facilities at Penn State University College of Medicine.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Pennsylvania State University College of Medicine.
1. Selection of animals for the study
<ol>
	<li>Maintain 18 healthy 10&#8722;12-week old male NOD.Cg-Rag1tm1Mom&#160;Il2rgtm1Wjl/SzJ (NRG) mice in a barrier environment under pathogen-free conditions. 
	NOTE: The common source for the right mouse strain for the study is peer-reviewed pub...

<ol>
	<li>Lowenberg, B., Rowe, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+the+review+series+on+advances+in+acute+myeloid+leukemia+(AML).">Introduction to the review series on advances in acute myeloid leukemia (AML).</a> Blood. 127 (1), 1 (2016).</li><li>De Kouchkovsky, I., Abdul-Hay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myeloid+leukemia:+a+comprehensive+review+and+2016+update.">Acute myeloid leukemia: a comprehensive review and 2016 update.</a> Blood Cancer Journal. 6 (7), 441 (2016).</li><li>. Cancer Stat Facts: Leukemia - Acute Myeloid Leukemia (AML) Available from: <a target="_blank" href="https://seer.cancer.gov/statfacts/html/amyl.html">https://seer.cancer.gov/statfacts/html/amyl.html</a> (2019)</li><li>Yang, X., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+therapy+for+acute+myeloid+leukemia.">Precision therapy for acute myeloid leukemia.</a> Journal of Hematology &amp; Oncology. 11 (1), 3 (2018).</li><li>Zhang, Z., Tang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+metabolism+in+drug+discovery+and+development.">Drug metabolism in drug discovery and development.</a> Acta Pharmaceutica Sinica B. 8 (5), 721-732 (2018).</li><li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nature Reviews: Drug Discovery. 5 (9), 741-754 (2006).</li><li>Denayer, T., St&#246;hr, T., Van Roy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+translational+medicine:+Validation+and+prediction.">Animal models in translational medicine: Validation and prediction.</a> New Horizons in Translational Medicine. 2 (1), 5-11 (2014).</li><li>Kuzu, O. F., Nguyen, F. D., Noory, M. A., Sharma, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+state+of+animal+(mouse)+modeling+in+melanoma+research.">Current state of animal (mouse) modeling in melanoma research.</a> Cancer Growth and Metastasis. 8, 81-94 (2015).</li><li>Yada, E., Wada, S., Yoshida, S., Sasada, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+patient-derived+xenograft+mouse+models+in+cancer+research+and+treatment.">Use of patient-derived xenograft mouse models in cancer research and treatment.</a> Future Science OA. 4 (3), 271 (2017).</li><li>Cheung, S. T., Shakibakho, S., So, E. Y., Mui, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfecting+RAW264.+7+cells+with+a+luciferase+reporter+gene.">Transfecting RAW264. 7 cells with a luciferase reporter gene.</a> Journal of Visualized Experiments. (100), e52807 (2015).</li><li>Li, J., Low-Nam, S., Garg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Luciferase-expressing+Tumor+Cell+Lines.">Generation of Luciferase-expressing Tumor Cell Lines.</a> Bio-protocol. 8 (8), 2817 (2018).</li><li>Barve, 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=Comparative+utility+of+NRG+and+NRGS+mice+for+the+study+of+normal+hematopoiesis,+leukemogenesis,+and+therapeutic+response.">Comparative utility of NRG and NRGS mice for the study of normal hematopoiesis, leukemogenesis, and therapeutic response.</a> Experimental Hematology. 67, 18-31 (2018).</li><li>Lee, E. 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=Efficacy+of+an+Fc-modified+anti-CD123+antibody+(CSL362)+combined+with+chemotherapy+in+xenograft+models+of+acute+myelogenous+leukemia+in+immunodeficient+mice.">Efficacy of an Fc-modified anti-CD123 antibody (CSL362) combined with chemotherapy in xenograft models of acute myelogenous leukemia in immunodeficient mice.</a> Haematologica. 100 (7), 914-926 (2015).</li><li>Verhagen, H. J., 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=IGFBP7+Induces+Differentiation+and+Loss+of+Survival+of+Human+Acute+Myeloid+Leukemia+Stem+Cells+without+Affecting+Normal+Hematopoiesis.">IGFBP7 Induces Differentiation and Loss of Survival of Human Acute Myeloid Leukemia Stem Cells without Affecting Normal Hematopoiesis.</a> Cell Reports. 25 (11), 3021-3035 (2018).</li><li>Xie, C., 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=Panobinostat+enhances+cytarabine+and+daunorubicin+sensitivities+in+AML+cells+through+suppressing+the+expression+of+BRCA1,+CHK1,+and+Rad51.">Panobinostat enhances cytarabine and daunorubicin sensitivities in AML cells through suppressing the expression of BRCA1, CHK1, and Rad51.</a> PLoS One. 8 (11), 79106 (2013).</li><li>Hengst, J. 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=SKI-178:+A+multitargeted+inhibitor+of+sphingosine+kinase+and+microtubule+dynamics+demonstrating+therapeutic+efficacy+in+acute+myeloid+leukemia+models.">SKI-178: A multitargeted inhibitor of sphingosine kinase and microtubule dynamics demonstrating therapeutic efficacy in acute myeloid leukemia models.</a> Cancer Translational Medicine. 3 (4), 109 (2017).</li><li>Song, C., 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=Targeting+casein+kinase+II+restores+Ikaros+tumor+suppressor+activity+and+demonstrates+therapeutic+efficacy+in+high-risk+leukemia.">Targeting casein kinase II restores Ikaros tumor suppressor activity and demonstrates therapeutic efficacy in high-risk leukemia.</a> Blood. 126 (15), 1813-1822 (2015).</li><li>Wunderlich, 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=Improved+chemotherapy+modeling+with+RAG-based+immune+deficient+mice.">Improved chemotherapy modeling with RAG-based immune deficient mice.</a> PLoS One. 14 (11), 0225532 (2019).</li></ol>

Critical steps in the protocol 
Characterization of the mouse model: Each cell line and primary cell-derived murine xenograft has a unique disease and host-specific factors that influence the time and characteristics of engraftment. Time to engraftment is usually defined as time taken for the bone marrow to have 25% blast cells or have bioluminescence signaling at least two logs higher than background or non-tumor bearing mice. It is critical to characterize the mouse model prior to starting an expe...

Acute myeloid leukemia (AML) is a clonal disorder arising from a malignantly transformed multipotent hematopoietic stem cell that acquires consecutive genomic alterations, eventually advancing into clinically overt disease. It is a highly complex disease with significant genetic, epigenetic, and phenotypic heterogeneity1. The uncontrolled proliferation and impaired differentiation of myeloid precursor cells (i.e., blasts) is one of the hallmarks of AML, leading to anemia, thrombocytopenia, and eventually death2. According to the American Cancer Society, in 2019, ~21,450 new cases of AML will be diagnosed, and ~10,920 people will succumb to the disease3. Standard therapeutic options include cytarabine-based chemotherapy and hematopoietic stem cell transplantation (HSCT). The 5-year overall survival (OS) of patients younger than 60 years old is around 40%, and for those older than 60 years it is only 10&#8722;20%4.
Novel drug discovery and drug development is a formidable challenge for the scientific community as well as the pharmaceutical industry. On average, the development of a novel drug costs ~$2.6 billion and takes over 10 years5. Drug discovery for anticancer drugs is an inefficient and cumbersome process with 89% of drugs failing in preclinical testing to gain FDA approval6. Flawed preclinical research is one of the reasons for drug failures7. Although multiple models of cell culture and in&#160;vitro&#160;studies&#160;are&#160;useful&#160;and&#160;important&#160;for testing potential therapies, the drawback of cell line models&#160;is that the synthetic nature of their culture conditions means they do not necessarily reflect the behavior of the original cancer cells in patients8. Also, it is impossible to fully recapitulate the complexity of the whole organism and the bone marrow microenvironment in cell culture8. As a result, cell line-derived xenograft tumor mouse models were&#160;generated&#160;through&#160;the transplantation&#160;of&#160;well-established cancer&#160;cell lines into immunocompromised mice. The major advantage of the cell line-derived xenograft mouse models is that they more closely simulate the tumor&#8217;s microenvironment and pathophysiological conditions9.
Here, we provide a comprehensive protocol explaining the steps to generate leukemia xenograft mouse models using a stable AML cell line labeled with a luciferase reporter (U937-Luc-tdTomato). Details of lentiviral transduction of the leukemia cell line will not be explained10,11. We also describe a detailed protocol to monitor leukemia progression using bioluminescence imaging (BLI). Statistical analysis and reporting of the differences observed in leukemia progression in mice treated with control, with a single standard of care drug (i.e., cytarabine or daunorubicin), or a combination of both, will be detailed. The&#160;first&#160;part of this protocol (sections 1, 2, and 3) are devoted to selection of the mouse strain and generation of the xenograft model, where we describe the transplantation procedure followed by imaging, randomization, and drug treatment of mice. Later, we detail steps to collect leukemia cells and perform flow cytometric analysis using antibodies directed against intracellular and surface hematopoietic markers to determine their phenotype. Also included is the Wright-Giemsa staining of bone marrow and spleen cells, which was done to show blast number or structural differences following drug treatment.
This protocol is robust and highly reproducible. The data shown here will aid investigators in testing the efficacy of novel therapeutic drugs. The immunocompromised RAG deleted mouse model used here is an established model for these kinds of studies, where tumor burden and survival rates can be monitored during the treatment regimen. Additionally, this method can be used to provide information on the proliferation and survival of leukemic cells and other hematopoietic cell populations throughout diagnosis.

<table border="0" cellpadding="0" cellspacing="0" style="width:845px;" width="844">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">1 mL Syringe</td>
			<td style="width:160px;">Fisher Scientific</td>
			<td style="width:304px;">309659</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 inch short bevel 20 g needle</td>
			<td>Fisher Scientific</td>
			<td>305179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>02-682-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">13 mm Single Ring slide</td>
			<td>Fisherbrand</td>
			<td>22-037-241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Polypropylene Conical Tube</td>
			<td>FALCON</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X RBC Lysis Buffer</td>
			<td>Fisher Scientific</td>
			<td>501129751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 mL microcentrifgue tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4.5 inch blunt/straight tip scissors</td>
			<td>Fisher Scientific</td>
			<td>28251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4.5 inch serated straight forceps</td>
			<td>VWR</td>
			<td>82027-440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL, 12x75 mm round bottom test tubes (flow tubes)</td>
			<td>Corning</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5/8 inch sterile 25 g needle</td>
			<td>Fisher Scientific</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well Non Treated Cell Culture Plate</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7AAD</td>
			<td>Biolegend</td>
			<td>420404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Prep</td>
			<td>COVIDIEN</td>
			<td>6818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil</td>
			<td>VWR</td>
			<td>89107-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoFlow IR Water-Jacketed CO2 Incubator</td>
			<td>NUAIRE</td>
			<td>Model no. NU-8700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood Collection</td>
			<td>RAM Scientific</td>
			<td>76011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Brilliant Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>563794</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BV421-human CD33</td>
			<td>Biolegend</td>
			<td>366622</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">BV-650 mouse CD45</td>
			<td>BD Biosciences</td>
			<td>563410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Analyzer</td>
			<td>EMD Millipore Corparation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer</td>
			<td>FALCON</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge machine</td>
			<td>BECKMAN COULTER</td>
			<td>605168-AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Count &#38; Viability Kit</td>
			<td>EMD Millipore Corparation</td>
			<td>MCH100102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytocentrifuge</td>
			<td>Fisher Scientific</td>
			<td>A78300003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytoclip Slide Clip</td>
			<td>Fisher Scientific</td>
			<td>59-910-052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide (DMSO)</td>
			<td>EMD Millipore Corparation</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable Centrifuge Tube</td>
			<td>Fisher Scientific</td>
			<td>05-539-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Luciferin-Sodium Salt</td>
			<td>GoldBio</td>
			<td>LUCNA-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS buffer (PBS with 2% FBS (Heat-inactivated))</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Cards for Cytospin</td>
			<td>Fisher Scientific</td>
			<td>22-030410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC-human CD45</td>
			<td>Biolegend</td>
			<td>304014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixable Viability Dye</td>
			<td>Thermo Fischer</td>
			<td>65-0864-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer</td>
			<td>Biolegend</td>
			<td>420801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometry analysis software</td>
			<td>FlowJo, LLC</td>
			<td>Version 10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Freezing Medium (90% FBS + 10% DMSO)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graduated Tips</td>
			<td>USASCIENTIFIC</td>
			<td style="width:304px;">10/20 &#181;L (1110-3700), 200 &#181;L (1111-1700), 
			and 1000 &#181;L (1111-2720)</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat Inactivated Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>H17112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">In Vivo Imaging system</td>
			<td>Perkin Elmer</td>
			<td>CLS136331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringes</td>
			<td>BD</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inverted Microscope</td>
			<td>Olympus</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td>VEDCO</td>
			<td>NDC 50989-150-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LABGARD CLASS II TYPE A2 BIOLOGICAL SAFETY CABINET</td>
			<td>NUAIRE</td>
			<td>Model no. NU-425-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image Software &#8211; IVIS Lumina Series</td>
			<td>Perkin Elmer</td>
			<td>128110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Flow CO2 Regulator</td>
			<td>E-Z Systems</td>
			<td>EP-1305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MB-10 tablets, sterilant</td>
			<td>Quip Laboratories</td>
			<td>MBTAB75BX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro cover glass</td>
			<td>VWR</td>
			<td>48366 205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting medium</td>
			<td>Fisher Scientific</td>
			<td>SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-human Fc receptor antibody</td>
			<td>BD Biosciences</td>
			<td>564220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse cage lid for euthanasia</td>
			<td>E-Z Systems</td>
			<td>E-20028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG)</td>
			<td>Jackson laboratory</td>
			<td>7799</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ntracellular Staining Permeabilization Wash Buffer (10X)</td>
			<td>Biolegend</td>
			<td>421002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-anti-BTK</td>
			<td>Biolegend</td>
			<td>558528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-anti-pSTAT3-tyr705</td>
			<td>Biolegend</td>
			<td>651004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilllin Streptomycin Solution, 100X</td>
			<td>CORING</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate-Buffered Saline (PBS)</td>
			<td>CORNIG cellgro</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipet controller</td>
			<td>DRUMMOND Scientific</td>
			<td>109883</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Pipette</td>
			<td>Eppendord Research</td>
			<td style="width:304px;">2.5 (O24694B), 10 (O31418B), 20 (O24694B), 
			100 (O337778), 200 (O26279B) and 1000 (O40665B)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti-mouse CD16/32 antibody</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RBC Lysis Buffer (10X)</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Refrigerated Centrifuge</td>
			<td>NuAire</td>
			<td>NU-C200R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reusable sample chamber</td>
			<td>Fisher Scientific</td>
			<td>5991040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td>CORING</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipet</td>
			<td>FALCON</td>
			<td>5mL (357543), 10mL (357551), 25mL (357535)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Styptic Powder</td>
			<td>Fisher Scientific</td>
			<td>NC1577028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tailveiner for mouse</td>
			<td>Agnthos</td>
			<td>TV-150</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Trypan Blue Solution</td>
			<td>Corning</td>
			<td>25900051</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Wipes</td>
			<td>Fisher Scientific</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Wright-Giemsa Stain</td>
			<td>VWR</td>
			<td>10143-106</td>
			<td></td>
		</tr>
	</tbody>
</table>

a xenograft mouse model to assess efficacy of therapeutic agents for human acute leukemia

Preclinical evaluation of therapeutic agents using an appropriate animal model is a critical step and a requirement for selecting drugs worth testing in humans. Therapeutic agents such as small molecule inhibitors, biological agents, immune checkpoint inhibitors, and immunotherapy each have unique mechanisms of action and call for careful selection of in vivo systems in which their efficacy can be tested. The purpose of this article is to describe in detail development of one such leukemia xenograft model for testing the therapeutic efficacy of novel agents. Using an immunocompromised (NRG) murine model that lacks B, T, and NK cells helps engraftment of transplanted leukemia cells and provides an acceptable microenvironment to study the therapeutic efficacy of small molecule inhibitors and some biological agents. This article describes the development of leukemia murine xenografts for in vivo drug testing using an acute myeloid leukemia (AML) cell line murine model treated with the cytotoxic drugs daunorubicin and cytarabine as an example. Treatment response can be assessed during therapy using several noninvasive and minimally invasive methods. Bioluminescence imaging can be used to measure leukemia burden over time when luciferase prelabeled leukemia cells are used to develop xenografts. Peripheral blood count analysis provides vital information about side effects such as myelosuppression (e.g., cytopenia) and therapeutic effect (e.g., blast count or differentiation). These techniques help track differences in the development of leukemia or decrease in tumor burden at various time points during the drug treatment without scarifying the study animals. Secondary methods such as immunophenotyping using flow cytometry are applied to confirm differences in the leukemia burden among treated and untreated groups. The methods described here can be tailored and used for developing xenografts of other types of leukemia (e.g., acute lymphoblastic leukemia).

We developed a model to study the standard of care chemotherapeutic regimen for AML in a mouse model. Luciferase and tdTomato-expressing U937 cells were cultured to allow a few passages. The luciferase activity of the cells was checked using the BLI system and found to be highly active (Figure 1A). Cells were observed under a fluorescence microscope to confirm the tdTomato expression (Figure 1B). NRG mice were injected intravenously with U937-Luc-tdTomato cells,...

Mouse (Mus Musculus) models are being widely used to develop xenografts using human leukemia cells. These models provide a comparable biological system to study drug efficacy, pharmacodynamics, and pharmacokinetics. Modeling acute myeloid leukemia in immunocompromised mice is described in detail using the U937 cell line xenograft as an example.

A Xenograft Mouse Model to Assess Efficacy of Therapeutic Agents for Human Acute Leukemia

Preclinical evaluation of therapeutic agents using an appropriate animal model is a critical step and a requirement for selecting drugs worth testing in ...