Name: intra-peritoneal transplantation for generating acute myeloid leukemia in mice
Uploaded: 2023-01-06T14:10:00
Description: Watch this Scientific Journal Video about Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice at JoVE.com

The authors thank Huck Institute&#39;s Flow Cytometry Core Facility and the Histopathology Core Facility of the Animal Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, for providing timely technical support. This work was supported by grants from the American Institute for Cancer Research (KSP), Penn State College of Agricultural Sciences, Penn State Cancer Institute, USDA-NIFA project 4771, Accession number 00000005 to K.S.P. and R.F.P.

All experiments were preapproved by the Institutional Animal Care and Use Committee at the Pennsylvania State University.
1. Preparation of buffers and reagents
<ol>
	<li>Prepare ampicillin supplemented (AP) LB agar plates (sterile 10 cm plates). To do this, dissolve 10 g of LB broth with agar in 400 mL of distilled water, stir, and bring the volume up to 500 mL. Sterilize the solution by autoclaving, then allow the solution to cool down, add 0.5 mL of ampicillin (stock: 150...

<ol>
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These above-described studies provide supportive evidence that the transplantation of Lin- cells is comparable to LSK cells in the generation of 1&#730; murine AML. In addition, the current data also shows that i.p. injection is an efficient and convenient method to establish murine AML compared to intravenous (or r.o.) injection.
In addition to LSK cells, other populations such as granulocyte-monocyte progenitor (GMP), common lymphoid progenitor (CLP), and common myeloid progenitor...

Acute myeloid leukemia (AML) is a type of hematologic malignancy of diverse etiology with poor prognosis1. The generation of AML animal models lays the foundation for the understanding of its complex variations and pathobiology in an effort to discover novel therapies2. Leukemogenesis in mice involves the transplantation of donor cells expressing fusion oncoproteins, including fusions involving the mixed lineage leukemia (MLL) gene to potently induce AML, to mimic the disease in humans3. Various cellular origins of donor cells have been reported in the transplantation of MLL gene-associated AML4, with very little being known about the cells responsible for the disease origin.
Multiple routes have been developed for transplantation in mice; rather than an intra-femoral injection, which directly introduces mutant donor cells into bone marrow5, an intravenous injection that utilizes the venous sinus plexus, tail vein, and jugular vein has been widely used to generate murine AML models6,7,8,9. In the case of retro-orbital (r.o.) injection, various inherent disadvantages, such as volume limitation, high technical demand, few chances for repeated attempts or error, and potential ocular injuries, have been major stumbling blocks with limited or no viable alternatives7. Tail vein injection can have similar problems besides local injuries; to facilitate the procedure, mice often need to be warmed up to dilate their tail veins10. It is also hard to locate the tail vein without an additional light source, particularly in the C57BL/6 strain of mice. For jugular vein injection, research personnel require sufficient training to locate the vein and limit possible complications. In addition, both venous sinus and jugular vein injections need to be performed under anesthesia, which adds another level of complexity. Thus, it is tempting to explore new routes for transplantation to facilitate the establishment of AML murine models.
Intra-peritoneal (i.p.) injection is commonly used to administer drugs, dyes, and anesthetics11,12,13,14,15; it has also been used to introduce hematopoietic cells for ectopic hematopoiesis16 and to transplant bone marrow-derived mesenchymal stem cells in various mouse models17,18,19,20,21. However, it has been infrequently used to establish hematopoietic malignancies in mice, particularly to study AML disease progression.
The present study describes the feasibility of i.p. injection in the generation of AML mouse models, in addition to comparing the transplantation efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor cells. These findings provide a simple and efficient way to generate experimental models of AML and related myeloid leukemias. Such a method has the potential to further our understanding of the disease mechanisms as well as provide a relatively easy model to test experimental therapies.

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	<tbody>
		<tr>
			<td>a-Select competent cells&#160;</td>
			<td>Bioline</td>
			<td>BIO-85027</td>
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			<td>Ammonium chloride (NH4Cl)</td>
			<td>Sigma Aldrich</td>
			<td>Cat# A-9434</td>
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			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>Cat# A0797</td>
			<td></td>
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			<td>Bovine Serum Albumin (BSA), Fraction V&#8212;Low-Endotoxin Grade</td>
			<td>Gemini bio-products</td>
			<td>Cat# 700-102P</td>
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			<td>Ciprofloxacin HCl</td>
			<td>GoldBio.com</td>
			<td>Cat# C-861-100</td>
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			<td>DMEM, high glucose, no glutamine</td>
			<td>Gibco</td>
			<td>Cat# 11960-044</td>
			<td></td>
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			<td>Dulbecco&#8217;s Phosphate-Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>Cat# 21-031-CV</td>
			<td></td>
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			<td>EDTA, Disodium Salt (EDTA-2Na), Dihydrate, Molecular Biology Grade</td>
			<td>Calbiochem</td>
			<td>Cat# 324503</td>
			<td></td>
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			<td>Fetal Bovine Serum - Premium Select</td>
			<td>Atlanta Biologicals</td>
			<td>Cat# S11550</td>
			<td></td>
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			<td>Holo-transferrin, bovine</td>
			<td>Sigma Aldrich</td>
			<td>Cat# T1283</td>
			<td></td>
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			<td>Insulin solution human</td>
			<td>Sigma</td>
			<td>Cat# I-9278</td>
			<td></td>
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			<td>Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>Gibco</td>
			<td>Cat# 12440-053</td>
			<td></td>
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			<td>L-glutamine 200 mM (100&#215;) solution</td>
			<td>HyClone, Gelifesciences</td>
			<td>Cat# SH30034.01</td>
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			<td>LB broth, Lennox</td>
			<td>NEOGEN</td>
			<td>Cat #: 7290A</td>
			<td></td>
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			<td>LB Broth with agar (Miller)</td>
			<td>Sigma Aldrich</td>
			<td>Cat# L-3147</td>
			<td></td>
		</tr>
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			<td>Mouse anti-mouse CD45.1 (FITC)</td>
			<td>Miltenyi Biotec</td>
			<td>Cat# 130-124-211</td>
			<td></td>
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			<td>Mouse Interleukin-3 (IL-3)</td>
			<td>Gemini bio-products</td>
			<td>Cat# 300-324P</td>
			<td></td>
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			<td>Mouse Interleukin-6 (IL-6)</td>
			<td>Gemini bio-products</td>
			<td>Cat# 300-327P</td>
			<td></td>
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			<td>Mouse Stem Cell Factor (SCF)</td>
			<td>Gemini bio-products</td>
			<td>Cat# 300-348P</td>
			<td></td>
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			<td>Penicillin-Streptomycin Solution, 100x</td>
			<td>Corning</td>
			<td>Cat# 30-002-CI</td>
			<td></td>
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			<td>Phenix-Eco (pECO) cells</td>
			<td>ATCC</td>
			<td>CRL-3214</td>
			<td></td>
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			<td>Potassium Bicarbonate (KHCO3), Granular</td>
			<td>JT. Baker</td>
			<td>Cat# 2940-01</td>
			<td></td>
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			<td>Rat anti-mouse CD117 (c-kit) (APC)</td>
			<td>BioLegend&#160;</td>
			<td>Cat # 105812</td>
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		<tr>
			<td>Rat anti-mouse Ly-6A/E (Sca-1) (PE-Cy7)</td>
			<td>BD Pharmingen</td>
			<td>Cat# 558162</td>
			<td></td>
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			<td>Recombinant Murine Flt3-Ligand</td>
			<td>Pepro Tech, INC.</td>
			<td>Cat# 250-31L</td>
			<td></td>
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			<td>RetroNectin Recombinant Human Fibronectin Fragment</td>
			<td>TaKaRa</td>
			<td>Cat# T100A</td>
			<td></td>
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			<td>TransIT-293 Reagent</td>
			<td>MirusBio</td>
			<td>Cat# MIR 2705</td>
			<td></td>
		</tr>
		<tr>
			<td>TRI Reagent</td>
			<td>Sigma Aldrich</td>
			<td>Cat# T9424</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>Cat # 15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>Cat# 25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol (BME)</td>
			<td>Sigma Aldrich</td>
			<td>Cat# M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial Assays&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit&#160;</td>
			<td>StemCell technologies</td>
			<td>Cat# 19856A</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Cat# 4368813</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa qPCR SuperMix</td>
			<td>Quanta Bio</td>
			<td>Cat# 95051-500</td>
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			<td>Plasmid Maxi Kit (25)</td>
			<td>Qiagen</td>
			<td>Cat#:12163</td>
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			<td>Animals</td>
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			<td>Ai14TdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td>Strain # 007914</td>
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			<td>CD45.1 C57BL6/J mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td>Strain # 002014</td>
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			<td>CD45.2 C57BL6/J mice&#160;</td>
			<td>Jackson Laboratory</td>
			<td>Strain # 000664</td>
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			<td>Instruments and Softwares</td>
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			<td>Adobe illustrator&#160;</td>
			<td>Version 25.2.3</td>
			<td></td>
			<td></td>
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		<tr>
			<td>BD accuri C6 flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
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			<td>FlowJo 10.8.0</td>
			<td>BD</td>
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			<td>GeneSys software program&#160;</td>
			<td>Version 1.5.7.0</td>
			<td></td>
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			<td>GraphPad Prism version 6&#160;</td>
			<td>GraphPad</td>
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			<td>Hemavet 950FS&#160;</td>
			<td>Drew Scientific</td>
			<td></td>
			<td></td>
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		<tr>
			<td>7300 Real time PCR system</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syngene G:BOX Chemi XR5 Chemiluminescence Fluorescence Imaging</td>
			<td>G:Box Chemi</td>
			<td></td>
			<td></td>
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</table>

intra-peritoneal transplantation for generating acute myeloid leukemia in mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells (LSCs). An experimental AML rodent model to test therapies based on successfully transplanting these cells via retro-orbital injections in recipient mice is fraught with challenges. The aim of this study was to develop an easy, reliable, and consistent method to generate a robust murine model of AML using an intra-peritoneal route. In the present protocol, bone marrow cells were transduced with a retrovirus expressing human MLL-AF9 fusion oncoprotein. The efficiency of lineage negative (Lin-) and Lin-Sca-1+c-Kit+ (LSK) populations as donor LSCs in the development of primary AML was tested, and intra-peritoneal injection was adopted as a new method to generate AML. Comparison between intra-peritoneal and retro-orbital injections was done in serial transplantations to compare and contrast the two methods. Both Lin- and LSK&#160;cells transduced with human MLL-AF9 virus engrafted well in the bone marrow and spleen of recipients, leading to a full-blown AML. The intra-peritoneal injection of donor cells established AML in recipients upon serial transplantation, and the infiltration of AML cells was detected in the blood, bone marrow, spleen, and liver of recipients by flow cytometry, qPCR, and histological analyses. Thus, intra-peritoneal injection is an efficient method of AML induction using serial transplantation of donor leukemic cells.

Comparison of the transplantation efficiency of murine AML cells using r.o. and i.p. routes of transplantation 
Previously, the establishment of 1&#730; AML was reported in recipient mice retro-orbitally transplanted with MLL-AF9-transduced LSK cells, and the transplantability of 1&#730; AML cells was demonstrated by serial transplantation30. The present study is the first to evaluate the possibility of using bone marrow Lin- cells to perform transplantation. The p...

Watch this Scientific Journal Video about Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice at JoVE.com

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

Here, intra-peritoneal injection of leukemia cells is utilized to establish and propagate acute myeloid leukemia (AML) in mice. This new method is effective in the serial transplantation of AML cells and can serve as an alternative for those who may experience difficulties and inconsistencies with intravenous injection in mice.

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

This protocol is significant because lin-negative cells and SK cells isolated from the same donor mice had no apparent difference in the generation of MLL-AF9 induced AML. Also, IP injection of leukemic cells can successfully develop a mouse model of AML. Compared to conventional transplantation methods, this technique is more convenient and less expensive.To begin, sterilize the 8 to 10-week-old CD45.1 female C57BL/6J mouse with 70%ethanol. Place the mouse onto a sterile surgical pad on a styrofoam board and pin the legs through the mouse paw pads. Cut the skin above the abdominal cavity at the midline and widen the subcutaneous space toward the hind legs with sharp-end sterile scissors.Extend the incision from the abdominal midline down to the ankles and widen the subcutaneous space below the hind legs with the blades of sharp-end scissors. Cut the achilles tendon with sharp-end scissors. Hold the tendon using forceps with teeth and cut the other end attached to the femur to remove the gastrocnemius muscle.Cut the quadriceps tendon attached to the knee with sharp-end scissors. Hold the tendon and cut the muscle head attached to the femur to remove the gastrocnemius muscle. Cut the other muscles surrounding the femur at the end attached to the tibia.Next, cut the ankle with sharp-end scissors, ensuring that the tibia remains intact. Hold the distal end of the femur and cut the hip joint with sharp-end scissors, ensuring that the femoral head remains intact. Transfer the tibias and femurs to a flow buffer in a 15 milliliter tube.Separate the tibia and femur by breaking the knee by hand. Remove the patella, cartilage, and femoral condyles to expose the tibial plateau and distal femur. Remove the muscles using sterile gauze and soak the bones in a flow buffer.Cut the femoral neck and flush the bone marrow cells with flow buffer from both ends of the femur using a 10 milliliter syringe with a 23 gauge needle. Next, cut the tibial malleolus and flush the bone marrow cells with flow buffer from both ends of the tibia. Disperse the cells by pipetting up and down using a 10 milliliter syringe with an 18 gauge needle.Centrifuge the single-cell suspension at four degrees Celsius and 400 G for three minutes. Discard the supernatant and resuspend the cells in five milliliters of red blood cell, or RBC, lysis buffer to lyse the RBCs for three minutes. Add five milliliters of flow buffer to stop the lysis.Then centrifuge the cell suspension and resuspend the pellet in five milliliters of flow buffer. Place a 70 micron cell strainer onto a 50 milliliter tube and pass the suspension through the strainer to collect the cells. Adjust the cell concentration with flow buffer to one times 10 to the eight per milliliter in round bottom polypropylene tubes.Select lineage-negative cells using a mouse hematopoietic cell isolation kit according to the manufacturer's instructions. Once done, centrifuge the sorted hematopoietic stem cells and unsorted lineage-negative cells and resuspend in three milliliters of 2X of cytokines, IMDM media, and three milliliters of viral supernatant in a retro-reductant coated dish. Incubate the dish in a humidified 5%carbon dioxide incubator at 37 degrees Celsius for 6 or 24 hours.After transduction, harvest the cells by centrifugation. If needed, use trypsin to collect the cells attached to the dish bottom. Discard the supernatant and resuspend the pellet in pre-warmed PBS.Inject cells into the primary recipient mouse retro-orbitally or intraperitoneally with a 27 gauge half needle. Monitor the mouse daily. After one month, collect blood weekly by retro-orbital bleeding to monitor leukocytosis by evaluating the complete blood count on a HEMAVET.Proptose the eye with the thumb and index finger, then penetrate the venous sinus plexus with a sterile hematocrit capillary tube through the inner canthus. Collect 20 to 25 microliters of blood into an EDTA blood collection tube and close the eyelids to stop the bleeding. Apply one drop of gentamycin sulfate ophthalmic solution to the eye.When the white blood cells reach four times 10 to the four cells per microliter, isolate the bone marrow cells by flushing the femurs and tibias with flow buffer, followed by RBC lysis as described earlier. Next, to harvest the splenocytes, place the mouse onto a sterile surgical pad on a styrofoam board and pin the legs through the mouse paw pads. Sterilize the mouse with 70%ethanol.Cut the skin and muscle at the midline to expose the abdominal cavity with sharp-end sterile scissors. Isolate the spleen and put it in a flow buffer in a 15 milliliter tube. Mesh the spleen through a 70 micron strainer in a six centimeter dish with three milliliters of flow buffer.Transfer the cells from the dish to a 15 milliliter tube, then centrifuge the single-cell suspension, discard the supernatant, and resuspend the pellet in five milliliters of RBC lysis buffer for three minutes. Add five milliliters of flow buffer to stop the lysis and centrifuge the cell suspension. Resuspend the pellet in five milliliters of flow buffer, mix and pass through a 70 micron cell strainer to collect cells into a 50 milliliter tube.Identify primary acute myeloid leukemia, or AML, cells by stain the splenocytes and bone marrow cells with FITC conjugated anti-mouse CD45.1 antibody and detecting on a flow cytometer. The cells are first gated on FSC-A FSC-H and FSC-A SSC-A to acquire singlets. The CD45.1 positive population is gated on the FL1 plot by comparing it to unstained cells.For secondary transplantation, resuspend CD45.1 AML splenic cells from primary retro-orbital recipients in PBS and inject them retro-orbitally into CD45.2 male mouse. The presence of aberrant leukocytosis and increased infiltration of leukemic cells in the bone marrow and spleen supports the feasibility of using bone marrow lineage-negative cells to generate primary AML. No significant differences were observed in primary recipients translated with LSK or lineage-negative cells.The AML cells spread in the abdominal cavity of primary recipients by mixed lineage leukemia or MLL-AF9 transduced lineage-negative cells. These results also confirmed the establishment of primary AML via intraperitoneal injection of MLL-AF9 transduced bone marrow lineage-negative cells in the form of leukocytosis and the presence of AML cells in the bone marrow and spleen of recipient mice. However, primary transplantation via intraperitoneal injection took longer to develop AML than via retro-orbital injection despite the equal number of donor cells.The secondary recipients showed leukocytosis and significant hepatosplenomegaly in less than one month post-transplantation. The AML cells were also detected in the peripheral blood, bone marrow, and spleen, as well as in the peritoneal cavity. The intraperitoneal injection of eight times 10 to the five AML cells achieved comparable engraftment to retro-orbital injection of eight times 10 to the five AML.The tertiary recipient mice acutely exhibited AML signs including leukocytosis and hepatosplenomegaly, the presence of leukemic cells in the blood, bone marrow, and spleen, histological observation of the femur and spleen further demonstrated the infiltration of leukemic cells. Compared to the secondary and tertiary transplantations, the tertiary transplantation progressed much faster than the secondary transplantation. For the current model, sorting LSK cells is unnecessary.Lin-negative cells can be transduced with AML virus for 6 hours or 24 hours. It won't affect the efficiency of the model establishment.

intra-peritoneal-transplantation-for-generating-acute-myeloid

Research

JoVE Journal

Cancer Research

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a &#39;molecular timeline&#39; since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to the recent FDA approval of several CD19-targeted CART (CART19) cell therapies. However, CART cell therapy is associated with a unique set of toxicities that carry their own morbidity and mortality. This includes cytokine release syndrome (CRS) and neuroinflammation (NI). The use of preclinical mouse models has been crucial in the research and development of CART technology for assessing both CART efficacy and CART toxicity. The available preclinical models to test this adoptive cellular immunotherapy include syngeneic, xenograft, transgenic, and humanized mouse models. There is no single model that seamlessly mirrors the human immune system, and each model has strengths and weaknesses. This methods paper aims to describe a patient-derived xenograft model using leukemic blasts from patients with acute lymphoblastic leukemia as a strategy to assess CART19-associated toxicities, CRS, and NI. This model has been shown to recapitulate CART19-associated toxicities as well as therapeutic efficacy as seen in the clinic.

Here, we describe a protocol in which an acute lymphoblastic leukemia patient-derived xenograft model is used as a strategy to assess and monitor CD19-targeted chimeric antigen receptor T cell-associated toxicities.

Chimeric antigen receptor T (CART) cell therapy has emerged as a powerful tool for the treatment of multiple types of CD19+ malignancies, which has led to ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...