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>
			<td></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>
			<td></td>
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		<tr>
			<td>FlowJo 10.8.0</td>
			<td>BD</td>
			<td></td>
			<td></td>
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			<td>GeneSys software program&#160;</td>
			<td>Version 1.5.7.0</td>
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		<tr>
			<td>GraphPad Prism version 6&#160;</td>
			<td>GraphPad</td>
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		<tr>
			<td>Hemavet 950FS&#160;</td>
			<td>Drew Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<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.

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