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.

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The authors declare no conflict of interest.

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 (CMP) have been reported to be substituted as donor cells in the generation of 1&#730; MLL-AF9-induced AML with various incubation durations31,32, although quantitative comparison is lacking to suggest the optimal choice. In the current study, Lin- and LSK isolated from the same donor mice had no apparent difference in the generation of 1&#730; MLL-AF9-induced AML, given that transduced myeloid progenitor cells (Sca-1-) were able to initiate AML33. Considering the expense and time required for flow cytometry-based sorting of LSK population, these studies support the use of the Lin- population as the donor cells for 1&#730; transplantation. Thus, the steps from 4.19 to 4.23 in this protocol are not necessary but optional, and do not affect the final result.
In terms of the amount of donor mice in 1&#730; transplantation, one donor was able to provide ~1.0 to 1.5 x 105 LSK cells, and one recipient transplanted with this amount of LSK cells developed 1&#730; AML in about 4 months. However, increasing the number of donors to build up LSK cells up to ~3 x 105 cells per recipient can shorten the incubation duration to ~70 days. Thus, two donors should be enough to rapidly generate 1&#730; AML. This principle also applies to Lin- cells, wherein ~1.0 to 2.5 x 106 Lin- cells can be isolated from each donor mouse. However, when donor cell density was increased, the turnaround time of leukemogenesis was considerably shorter. These data should be taken into consideration when deciding the number of donor mice in step 4.1.
Compared to r.o. injection, i.p. injection took about ~20 more days to develop the full-blown 1&#730; AML, however this limitation was overcome by increasing the donor cells for i.p. injection. Despite such an apparent difference, both methods showed similar engraftment rate (&#62;80%) in the bone marrow and spleen at the endpoint, indicating the general applicability of i.p. injection in 1&#730; transplantation in AML. The relatively long incubation period for 1&#730; transplantation, as well as its unpredictable disease progression in terms of inconsistency of latency in recipient mice, is a major limitation for controlled experiments, such as pharmacological interventions and mechanistic studies. Thus, it is recommended to use 1&#730; leukemic cells for transplantation in subsequent serial transplantations, typically 2&#730; transplantation (Figure 1). Since up to 3 x 108 leukemic cells in spleen (and 6 x 107 cells in bone marrow) can be generated from every single 1&#730; recipient at the endpoint, which would be sufficient to conduct 2&#730; transplantation on more than 300 mice, it is suggested to transplant as many cells as possible to fewer recipients to shorten the latency and increase the engraftment rate of 1&#730; transplantation, which is important for step 4.1 and step 5.2.
Given its consistent and short latency, 2&#730; transplantation can be utilized in multiple applications, including in vivo experiments mentioned above. Furthermore, i.p. injection also facilitates the procedure for abundant transplantation for inexperienced technicians. Compared to the r.o. injection route, which generally generates the AML in 3 weeks, i.p. injection method took slightly longer (~4 weeks) with the same number of donor cells to establish a full-blown AML. Even though it is relatively easier and more convenient, increasing the transplanted cell number or performing tertiary transplantation can also accelerate the progression of AML by i.p. injection.
In summary, the major limitation of this technique is the longer incubation time than intravenous injection with the same amount of donor cells in both 1&#730; and 2&#730; transplantations. However, it can be easily overcome by increasing the cell number to be transplanted. Apart from the obvious advantages, this technique also has some other applications. For example, the ability to use i.p. transplantation methods may also serve to routinely culture these cells without the need for expensive in vitro culture media. To extend the application of this technique in other hematological malignancies, such as lymphoid leukemia and chronic myeloid leukemia, and patient-derived xenografts, further studies need to be done in the future.

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.

<table><tbody><tr><td>a-Select competent cells&amp;nbsp;</td><td>Bioline</td><td>BIO-85027</td><td></td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Sigma Aldrich</td><td>Cat# A-9434</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>Cat# A0797</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA), Fraction V&amp;mdash;Low-Endotoxin Grade</td><td>Gemini bio-products</td><td>Cat# 700-102P</td><td></td></tr><tr><td>Ciprofloxacin HCl</td><td>GoldBio.com</td><td>Cat# C-861-100</td><td></td></tr><tr><td>DMEM, high glucose, no glutamine</td><td>Gibco</td><td>Cat# 11960-044</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Saline (PBS)</td><td>Corning</td><td>Cat# 21-031-CV</td><td></td></tr><tr><td>EDTA, Disodium Salt (EDTA-2Na), Dihydrate, Molecular Biology Grade</td><td>Calbiochem</td><td>Cat# 324503</td><td></td></tr><tr><td>Fetal Bovine Serum - Premium Select</td><td>Atlanta Biologicals</td><td>Cat# S11550</td><td></td></tr><tr><td>Holo-transferrin, bovine</td><td>Sigma Aldrich</td><td>Cat# T1283</td><td></td></tr><tr><td>Insulin solution human</td><td>Sigma</td><td>Cat# I-9278</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Gibco</td><td>Cat# 12440-053</td><td></td></tr><tr><td>L-glutamine 200 mM (100&amp;times;) solution</td><td>HyClone, Gelifesciences</td><td>Cat# SH30034.01</td><td></td></tr><tr><td>LB broth, Lennox</td><td>NEOGEN</td><td>Cat #: 7290A</td><td></td></tr><tr><td>LB Broth with agar (Miller)</td><td>Sigma Aldrich</td><td>Cat# L-3147</td><td></td></tr><tr><td>Mouse anti-mouse CD45.1 (FITC)</td><td>Miltenyi Biotec</td><td>Cat# 130-124-211</td><td></td></tr><tr><td>Mouse Interleukin-3 (IL-3)</td><td>Gemini bio-products</td><td>Cat# 300-324P</td><td></td></tr><tr><td>Mouse Interleukin-6 (IL-6)</td><td>Gemini bio-products</td><td>Cat# 300-327P</td><td></td></tr><tr><td>Mouse Stem Cell Factor (SCF)</td><td>Gemini bio-products</td><td>Cat# 300-348P</td><td></td></tr><tr><td>Penicillin-Streptomycin Solution, 100x</td><td>Corning</td><td>Cat# 30-002-CI</td><td></td></tr><tr><td>Phenix-Eco (pECO) cells</td><td>ATCC</td><td>CRL-3214</td><td></td></tr><tr><td>Potassium Bicarbonate (KHCO&lt;sub&gt;3&lt;/sub&gt;), Granular</td><td>JT. Baker</td><td>Cat# 2940-01</td><td></td></tr><tr><td>Rat anti-mouse CD117 (c-kit) (APC)</td><td>BioLegend&amp;nbsp;</td><td>Cat # 105812</td><td></td></tr><tr><td>Rat anti-mouse Ly-6A/E (Sca-1) (PE-Cy7)</td><td>BD Pharmingen</td><td>Cat# 558162</td><td></td></tr><tr><td>Recombinant Murine Flt3-Ligand</td><td>Pepro Tech, INC.</td><td>Cat# 250-31L</td><td></td></tr><tr><td>RetroNectin Recombinant Human Fibronectin Fragment</td><td>TaKaRa</td><td>Cat# T100A</td><td></td></tr><tr><td>&lt;em&gt;Trans&lt;/em&gt;IT-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>&amp;beta;-Mercaptoethanol (BME)</td><td>Sigma Aldrich</td><td>Cat# M3148</td><td></td></tr><tr><td>&lt;strong&gt;Commercial Assays&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit&amp;nbsp;</td><td>StemCell technologies</td><td>Cat# 19856A</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit&amp;nbsp;</td><td>Thermo Fisher&amp;nbsp;</td><td>Cat# 4368813</td><td></td></tr><tr><td>PerfeCTa qPCR SuperMix</td><td>Quanta Bio</td><td>Cat# 95051-500</td><td></td></tr><tr><td>Plasmid Maxi Kit (25)</td><td>Qiagen</td><td>Cat#:12163</td><td></td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Ai14&lt;sup&gt;TdTomato&lt;/sup&gt; &lt;/em&gt;mice</td><td>Jackson Laboratory</td><td>Strain # 007914</td><td></td></tr><tr><td>CD45.1 C57BL6/J mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>Strain # 002014</td><td></td></tr><tr><td>CD45.2 C57BL6/J mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>Strain # 000664</td><td></td></tr><tr><td>&lt;em&gt;Instruments and Softwares&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Adobe illustrator&amp;nbsp;</td><td>Version 25.2.3</td><td></td><td></td></tr><tr><td>BD accuri C6 flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>FlowJo 10.8.0</td><td>BD</td><td></td><td></td></tr><tr><td>GeneSys software program&amp;nbsp;</td><td>Version 1.5.7.0</td><td></td><td></td></tr><tr><td>GraphPad Prism version 6&amp;nbsp;</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Hemavet 950FS&amp;nbsp;</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></tr></tbody></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 presence of aberrant leukocytosis (Figure 2A) and increased infiltration of leukemic cells (CD45.1+) in the bone marrow and spleen (Figure 2B) support the feasibility of using bone marrow Lin- cells to generate 1&#730; AML. To compare the disease induction period, two donor mice per recipient were euthanized to isolate either LSK or Lin- cells. From the results, no significant differences were observed in 1&#730; recipients transplanted with LSK or Lin- cells (Figure 2C).
During the examination of metastasis of 1&#730; AML, it was discovered that the AML cells spread in the abdominal cavity of 1&#730; recipients by MLL-AF9-transduced Lin- cells (Supplementary Figure 1). This finding inspired the exploration to test whether i.p. injection could be adopted in the generation of murine AML, which might serve as a new and easier route of transplantation. Due to the success of using the bone marrow Lin- population as AML donor cells, current data confirmed the establishment of 1&#730; AML via i.p. injection of MLL-AF9-transduced bone marrow Lin- cells, as seen in the form of leukocytosis (Figure 2D) and the presence of AML cells (CD45.1+) in the bone marrow and spleen of recipient mice (Figure 2E). However, transplantation via i.p. injection took longer to develop AML than via r.o. injection, despite the recipients receiving an equal number of donor cells (Figure 2F). Besides, mice transplanted retro-orbitally with more Lin- cells (5.125 x 106 cells/mouse) in Figure 2F seemed to develop AML faster than those transplanted with fewer Lin- cells (3.69 x 106 cells/mouse) in Figure 2C.
Owing to the inherent inconsistency of incubation time in 1&#730; AML recipients (Figure 2F), an attempt was made to test the i.p. transplantation in 2&#730; recipients. For 2&#730; transplantation, i.p. injection was performed with 8 x 105 AML cells isolated from intra-peritoneally transplanted 1&#730; recipients. The 2&#730; recipients showed leukocytosis (Figure 2G) and significant hepatosplenomegaly (Figure 2H) in less than 1 month post transplantation, a clear advancement from 1&#730; transplantation. Consistent with this observation, AML cells (RFP-) were also detected in the peripheral blood, bone marrow, and spleen (Figure 2I), as well as in the peritoneal cavity (Supplementary&#160;Figure 1). Besides, the expression of oncogene KMT2A in the blood, spleen, and liver of 2&#730; recipients also confirmed the successful generation of AML (Figure 2J). Moreover, histological analysis further demonstrated the infiltration of leukemic cells in the spleen and liver of intra-peritoneally transplanted mice (Figure 2K). These data confirm that i.p. injection-generated 1&#730; AML cells are transplantable in 2&#730; recipients. Furthermore, i.p. injection of 8 x 105 AML cells per mouse into recipients achieved comparable engraftment to r.o. injection of 8 x 105 AML cells per mouse into recipients (Figure 2L).
One of the key features of LSCs is serial transplantability; thus, to further ascertain the establishment of AML in 2&#730; recipients, this protocol attempted to verify if AML cells from 2&#730; i.p. transplantation remained serially transplantable, as well as the feasibility of stem cell transplantation by i.p. injection in 3&#730; transplantation. AML cells isolated from either bone marrow (8 x 105 leukemic cells/mouse in 0.5 mL of PBS) or i.p. lavage (4 x 105 leukemic cells/mouse in 0.5 mL of PBS) of 2&#730; recipients were injected into 3&#730; recipients. Although being transplanted with donor cells generated from different sources, 3&#730; recipient mice acutely exhibited AML signs (within 3 and 4 weeks for bone marrow cells and i.p. cells, respectively), including leukocytosis (Figure 3A) and hepatosplenomegaly (Figure 3B), the presence of leukemic cells in the blood, bone marrow, and spleen (Figure 3C), and the expression of KMT2A (Figure 3D). Histological observation of the femur and spleen further demonstrated the infiltration of leukemic cells (Figure 3E). As a comparison, r.o. injection of leukemic splenocytes (4 x 105 cells) from 2&#730; recipients was also equally capable of engrafting and developing AML in 3&#730; recipients, characterized by leukocytosis (Supplementary&#160;Figure 2A), hepatosplenomegaly (Supplementary Figure 2B), and the infiltration of AML cells in the blood, bone marrow, and spleen (Supplementary Figure 2C).
Intra-peritoneal transplantation is effective even for 3&#730; transplantation of AML cells 
Compared to the 2&#730; and 3&#730; transplantations, a conclusion could easily be made that 3&#730; transplantation (Figure 3F) progressed much faster than 2&#730; transplantation (Figure 2L) for both i.p. and r.o. injections. Notably, i.p. injection of 4 x 105 AML cells/mouse developed AML later than r.o. injection of same number of AML cells for 6 days (Figure 3F), possibly indicating the time spent on the migration from the peritoneal cavity to the bone marrow niche. Interestingly, the high frequency of leukemic cells in the peritoneal cavity (Supplementary&#160;Figure S2) in the 3&#730; transplanted mice suggested that the peritoneal cavity may also provide the niche for the fast expansion of leukemic cells. Besides, the presence of leukemic cells in the peritoneal cavity of 1&#730; and 3&#730; recipients transplanted retro-orbitally indicated the mutual circulation of leukemic cells between the peritoneal cavity and the blood system, justifying the i.p. transplantation. Collectively, these findings validate the use of i.p. injection in serial transplantation of AML.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64834/64834fig02.jpg" /> 
Figure 2: Generation of murine AML model in 1&#730; and 2&#730; transplantation. (A) Complete blood count (CBC; 1 x 103 cells/&#181;L blood), WBC, neutrophil (NE), lymphocyte (LY), monocyte (MO), eosinophil (EO), and basophil (BA) profile of 1&#730; recipient mice transplanted retro-orbitally with MLL-AF9-transduced bone marrow Lin- cells (5.125 x 106 cells/mouse) by hemavet (n = 3). (B) Flow cytometric analysis of the frequency of AML cells (CD45.1+) in the bone marrow and spleen of 1&#730; CD45.2 recipient mice transplanted retro-orbitally with MLL-AF9-transduced bone marrow Lin- (5.125 x 106 cells/mouse) cells (n = 3). (C) Incubation duration of 1&#730; AML in recipients transplanted retro-orbitally with MLL-AF9-transduced bone marrow LSK cells (3.16 x 105 cells/mouse isolated from two donors, n = 4) or Lin- cells (3.69 x 106 cells/mouse isolated from two donors, n = 3). Each recipient received cells isolated from two donors. (D) CBC analysis of 1&#730; recipient mice transplanted intra-peritoneally with MLL-AF9-transduced bone marrow Lin- cells (5.125 x 106 cells/mouse) by hemavet (n = 4). (E) Flow cytometric analysis of the frequency of AML cells (CD45.1+) in the bone marrow and spleen of 1&#730; CD45.2 recipient mice transplanted intra-peritoneally with MLL-AF9-transduced bone marrow Lin- cells (5.125 x 106 cells/mouse, n = 3). (F) Incubation duration of 1&#730; AML in recipients transplanted retro-orbitally (n = 3) or intra-peritoneally (n = 3) with MLL-AF9-transduced bone marrow Lin- cells. Each recipient received the same amount of donor cells (5.125 x 106 cells/mouse). (G) CBC analysis of 2&#730; recipients transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1&#730; i.p. transplanted recipients by hemavet (n = 3). (H) Weights (mg) of the spleen and liver of 2&#730; recipient mice transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1&#730; i.p. transplanted recipients. The shaded areas represent the normal weight ranges of spleen and liver. Representative image of spleen and liver from 2&#730; recipient mice and healthy counterparts. (I) Flow cytometric analysis of the frequency of AML cells (RFP-) in the blood, bone marrow, and spleen of 2&#730; RFP+ recipient mice transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1&#730; i.p. transplanted recipients (n = 3). (J) Gene expression of KMT2A and 18S presented as a DNA band. RNAs were isolated from the blood, bone marrow, spleen, and liver of 2&#730; recipient mice transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1&#730; i.p. transplanted recipients. (K) Representative images of histological changes in the spleen (left panel) and liver (right panel) of 2&#730; recipients transplanted intra-peritoneally with AML cells (8 x 105 cells/mouse) isolated from the spleen of 1&#730; i.p. transplanted recipients and healthy counterparts. (L) Incubation duration of 2&#730; AML in recipients transplanted retro-orbitally (4 x 105/mouse, n = 9) or intra-peritoneally (8 x 105/mouse, n = 4) with AML cells isolated from 1&#730; r.o. and i.p. transplanted recipients, respectively. The results are mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64834/64834fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64834/64834fig03.jpg" /> 
Figure 3: 3&#730; transplantation of AML cells via i.p. transplantation. (A-E) 3&#730; RFP+ or CD45.2 recipient mice. Left: i.p. injection of AML cells (8 x 105 cells/mouse) from the bone marrow of 2&#730; recipients. Right: i.p. injection of AML cells (4 x 105 cells/mouse) from the peritoneal cavity (i.p.) of 2&#730; recipients (n = 3). (A) CBC analysis of 3&#730; recipient mice by hemavet. (B) Weights (mg) of the spleen and liver of 3&#730; recipient mice. The shaded areas represent the normal weight ranges of the spleen and liver. (C) Flow cytometric analysis of the frequency of AML cells (left: RFP-; right: CD45.1+) in the blood, bone marrow, and spleen of 3&#730; recipient mice. (D) Gene expression of KMT2A and 18S presented as a DNA band. RNAs were isolated from the blood, bone marrow, spleen, and liver of 3&#730; recipient mice. (E) Representative images of histological changes in the spleen (left panel) and femur (right panel) of 3&#730; recipient mice. (F) Incubation duration of 3&#730; AML in recipients transplanted retro-orbitally (4 x 105 cells/mouse, n = 3) or intra-peritoneally (4 x 105 cells/mouse, n = 3) with AML cells isolated from 2&#730; r.o. and i.p. transplanted recipients, respectively. The results are mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64834/64834fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Flow cytometric analysis of AML cell infiltration in the peritoneal cavity. Frequency of AML cells in the peritoneal lavage of 1&#730; recipients transplanted retro-orbitally (1&#730; RO, n = 2), 2&#730; recipients transplanted intra-peritoneally (2&#730; IP, n = 2), and 3&#730; recipients transplanted via i.p. injection with bone marrow AML cells (3&#730; BM-IP, n = 3) and i.p. AML cells (3&#730; IP-IP, n = 3), as well as 3&#730; recipients transplanted via r.o. injection of AML splenocytes (3&#730; SP-RO, n = 3). The results are mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64834/Supplementary Figure1.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: 3&#730; transplantation of AML splenocytes via r.o. injection. (A) CBC analysis of 3&#730; recipient mice retro-orbitally transplanted with AML cells (4 x 105 cells/mouse) from the spleen of 2&#730; recipient mice. (B) Weights (mg) of spleen and liver of 3&#730; retro-orbitally transplanted recipient mice. The shaded areas represent the normal weight ranges of the spleen and liver. (C) Frequency of AML cells (RFP-) in the blood, bone marrow, and spleen of 3&#730; retro-orbitally transplanted recipient mice (n = 3). The results are mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64834/Supplementary Figure2.pdf" target="_blank">Please click here to download this File.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.0K Views.  The Pennsylvania State University. 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. 

Video: Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

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 ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Medicine

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 ...

Busulfan as a Myelosuppressive Agent for Generating Stable High-level Bone Marrow Chimerism in Mice

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism&nbsp;>80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Immunology and Infection

Intrafemoral Injection for Bone Marrow Study in Live Mice

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. In murine models, this technique has been adapted to enhance the seeding efficiency of transplanted hematopoietic stem and progenitor cells (HSPCs). This paper describes a detailed step-by-step technical procedure of IF injection and the following bone marrow (BM) aspiration in mice that allows for serial characterization of cells present in the BM. This method enables the transplantation of valuable samples with low cell numbers that are particularly difficult to engraft by intravenous injection. This procedure facilitates the creation of xenografts that are critical for pathological analysis. While it is easier to access peripheral blood (PB), the cellular composition of PB does not reflect the BM, which is the niche for HSPCs. Therefore, procedures providing access to the BM compartment are essential for studying hematopoiesis. IF injection and serial BM aspiration, as described here, allow for the prospective retrieval and characterization of cells enriched in the BM, such as HSPCs, without sacrificing the mice.

This protocol describes the intrafemoral injection of a few hematopoietic or leukemic stem cells, including gene-edited cells, in murine xenograft models, which will not only enable the quick and safe transplantation of cells but also serial analyses of the bone marrow.

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Injecting Human HSPCs into the Femur of Sub-Lethally Irradiated Mice

Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. 
This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.

Author Spotlight: Exploring the Lifespan Dynamics of Healthy Human Hematopoiesis

Intrafemoral injections allow for the engraftment of a small number of hematopoietic stem and progenitor cells (HSPCs), by placing the cells directly in the bone marrow cavity. Here we describe an experimental protocol of intrafemoral injection of human HSPCs into immunodeficient mice.

Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell ...

Biology

Intraperitoneal Injection of Cells: A Method of Delivering Cancer Cells into the Peritoneal Adipose Tissue of Murine Model

Source: Krishnan, V. et al. <a href="https://www.jove.com/t/52721">Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose.</a> J. Vis. Exp. (2015)
In this video, we perform an intraperitoneal injection of ovarian cancer cell suspension in a mouse model to study the colonization of ovarian cancer cells in peritoneal adipose tissues and their subsequent metastasis.&#160;

Source: Krishnan, V. et al. Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose. J. Vis. Exp. 
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