Name: In Ovo Xenografting of Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells (PDX-ALL) (Video) | JoVE Journal
Uploaded: 2025-08-01T14:10:00.000+00:00
Duration: 6 min 48 s
Description: Learn in ovo xenografting of patient-derived acute lymphoblastic leukemia cells for cancer biology research. This rapid method enables preclinical drug screening and mechanistic studies using B-cell and T-cell lineages.

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5 minutes

We thank Dr. Andrew Kung at Columbia University for providing FUW-Luc-mCherry-puro. This work was supported by grants from CIHR (PJT-174983) to KYL and from the National Research Foundation of Korea (NRF; RS-2023-00284121) to KYL and M-YJ.

Experiments were performed based on the protocol approved by the Ethics Committee at the University of Calgary (REB15-1143; where informed consent was obtained from ALL subjects) and the Health Research Ethics Board of Alberta Cancer Committee (HREBA.CC-17-0108_REN8). Patients with ALL at diagnosis (before starting any therapy), who had circulating blasts (blasts not only in the marrow but also in the blood), were included in the study. The reagents and the equipment used are listed in the Table of Materials.
1. Egg incubation
<ol>
	<li>Procure fertilized chicken eggs. Obtain ~10% more eggs than needed to consider the number of eggs damaged during shipping or the cracking procedure.</li>
	<li>Store the fertilized eggs for up to two weeks at 12 &#176;C without losing viability.</li>
	<li>Transferthe eggs into a humidified (~50%-60%) rolling incubator at 39 &#176;C.</li>
	<li>Incubate the eggs in the incubator for 10dpf. Note that day 1 starts when the eggs are transferred into the incubator.</li>
</ol>
2. Isolation of acute lymphoblastic leukemia cells from blood samples from patients with ALL
<ol>
	<li>Isolate the cells as described previously14,15.</li>
	<li>Collect 10 mL of peripheral blood (or marrow) from B- and T-ALL patients into heparinized test tubes.</li>
	<li>Transfer the blood samples into a sterile 50 mL centrifuge tube and dilute thrice with 2 volumes of 1x phosphate-buffered saline (PBS) containing 137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, and 1.8 mM of KH2PO4.</li>
	<li>Overlay 1 volume of the diluted blood samples onto 1 volume of density gradient medium and centrifuge at room temperature without brake at 300 x g for 30 min.</li>
	<li>Collect the buffy coat layer of mononuclear cells (MNCs) from the interface and transfer it to a new sterile 15 mL tube using a sterile pipette.</li>
	<li>Add 10 mL of 1x PBS and centrifuge at 300 x g for 5 min to remove any remaining serum components (repeat this step twice).</li>
	<li>Add 2 mL of 0.8% NH4Cl to the cell pellet, vortex gently, incubate at room temperature for 5 min, and spin at 300 x g for 5 min to lyze red blood cells (If necessary, repeat this step).</li>
	<li>Wash the cells in 1x PBS as described in step 2.6.</li>
	<li>Resuspend the cells in RPMI 1640 containing 10% FBS and count the number of cells using a hemocytometer.</li>
	<li>Assess cell viability using the Trypan Blue Exclusion assay.</li>
	<li>Resuspend the cells in the freezing medium (10% DMSO in FBS), freeze them at -80 &#176;C, and store them in liquid nitrogen.</li>
</ol>
3. Amplification of 2nd-generation replication-incompetent lentivirus carrying mCherry
<ol>
	<li>Seed HEK293T human embryonic kidney cells at ~60%-70% confluency into a 6-well plate containing DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37 &#176;C in a humidified incubator with 5% CO2.</li>
	<li>Cotransfect the cells (1.5 x 106) with FUW-Luc-mCherry-puro transfer, pCMV&#916;r8.91 packaging and pVSV-G envelope vectors at a ratio of 3:2:1 using Lipofectamine 3000 for 2 days.</li>
	<li>Harvest the lentivirus-containing supernatant and filter through a 0.45 &#181;m filter.</li>
	<li>Transfer the cleared viral supernatant into centrifuge tubes, centrifuge at 20,000 x g for 2 h at 4 &#176;C, discard the supernatant, and resuspend the pellet in 100 &#181;L of PBS or serum-free medium.</li>
	<li>Measure the multiplicity of infection (MOI) using the qPCR Lentivirus Titer kit.</li>
</ol>
4. Labeling of continuously growing ALL cell lines and patient-derived ALL cells with mCherry
<ol>
	<li>Plate1 x 106 cells/mL of B-(SEM) and T-ALL (MOLT3)16,17cell lines and patient-derived B- or T-ALL cells in 6-well plates containing RPMI 1640 + 10% FBS.</li>
	<li>Infect the cells with 300 &#181;L of lentiviral preparation with a viral titer of 3.47 x 106 IU/mL to obtain MOI of 1 for 1 x 106 cells, mix gently, and incubate at 37 &#176;C with 5% CO2 for 2 days.</li>
	<li>Add 1 &#181;g/mL puromycin to the culture medium to select stably transduced cells for 2 days.</li>
	<li>Visualize mCherry-labeled cells with a fluorescence microscope.</li>
</ol>
5. In ovo xeno grafting
<ol>
	<li>On day 10 (see above the egg incubation section), examine the vasculature of the chick embryos under light to assess embryo viability. Then, wash eggs gently with 70% ethanol to sterilize the surface.</li>
	<li>Drillinto the air cell of the eggs to create a small (~2.0 cm in diameter) window using a hand drill.</li>
	<li>Seal with transparent adhesive tape and return to a 5% CO2 incubator.</li>
	<li>On day 11, inject 1 x 107 mCherry-labeled ALL cells into the blood vessels of the developing embryos using 34 G needles. Note that it turned out to be difficult to confirm whether ALL cells were injected into identical locations across different embryos because opening the window at the same locations is not guaranteed.</li>
	<li>Seal the windows with transparent adhesive tape, return to 5% CO2 incubator, and monitor the embryo viability daily.</li>
	<li>On day 15, dissect blood vessels out from embryos, place them on glass slides, and photograph successful xenografts using a dissecting microscope equipped with fluorescence capabilities. 
	NOTE: Beyond 15dpf (as feathers cover the entire body, and the albumen is nearly gone), immunohistochemistry needs to be performed to assess the changes in proliferation and colonization of ALL cells in the chick embryo vasculature.</li>
</ol>
6. Flow cytometry
<ol>
	<li>On day 15, collect blood from the chicken embryo vasculature using a sterile syringe with a 32 G needle, transfer it into a 1.5 mL sterile tube containing heparin, and centrifuge at 226 x g for 10 min at 4 &#176;C.</li>
	<li>Remove the supernatant (plasma) and resuspend the cell pellet in 1x PBS containing 1% BSA (bovine serum albumin).</li>
	<li>Perform step 2.7 and step 2.8 to lyse red blood cells.</li>
	<li>Label B-ALL cells with human FITC-CD10 and human PE-CD19 antibodies, and T-ALL cells with human FITC-CD4 and human PE-CD8 antibodies, respectively, at 4 &#176;C in the dark for 30 min.</li>
	<li>Wash the labeled cells twice with PBS containing 1% BSA, centrifuge at 226 x g for 5 min at 4 &#176;C, resuspend in 1x PBS, and analyze by flow cytometry.</li>
	<li>Set the forward scatter (FSC) and side scatter (SSC) voltages at 400 V and 360 V, respectively. Adjust the detector gains for the FITC and PE channels to 450-500 V and 400-450 V, respectively. 
	NOTE: Gating strategy: (a) light scatter gating (FSC&#160;vs. SSC): use FSC-A&#160;vs. SSC-A to identify a population with intermediate forward scatter (size) and low side scatter (granularity), which corresponds to the lymphoblast region. Note that this step helps exclude debris and more granular cells such as monocytes or granulocytes; (b) singlet selection (FSC-H&#160;vs. FSC-A): perform doublet discrimination by gating on FSC-H&#160;vs. FSC-A, ensuring only single-cell events are analyzed18.</li>
	<li>Quantify CD10/CD19+ B-ALL and CD4/CD8+ T-ALL cells by counting events in quadrant 2 (Q2; FITC/PE+) of FITC vs. PE plots18. Present means &#177; SEM of three independent experiments (n = 3).</li>
</ol>

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The authors declare no competing financial interests.

Three critical steps duringthe in ovo xenografting were identified to prevent microbial contamination and to maximize the survival of chicken embryos: i.e., (1) sterilizing the eggshell surface with 70% ethanol prior to making the window and (2) tight sealing of the eggshell window with Cellotape to prevent evaporation the content of chicken embryo during incubation, and (3) the use of the fine needle (e.g., 34 G) and injection of ALL cells in a minimal volume (e.g., 100 &#181;L) to ensure proper vascular delive...

The chick chorioallantoic membrane (CAM), starts to take shape at day 7 post-fertilization following incubation, and is complete by day 12. The CAM holds the limelight as a suitable in vivo model for angiogenesis1 and engrafting cancer cells2,3,4,5,6,7,8,9,10, particularly blood-related cancer cells11, as it is inherently immunodeficient and has well-developed vascularization. Moreover, the CAM is also a desirable system for investigating tumor invasion and metastasis as it carries a number of extracellular matrix proteins, including collagen, integrin &#945;V&#946;3, fibronectin, laminin, and matrix metalloproteinase-2 (MMP-2)10.
Acute lymphoblastic leukemia (ALL) is a cancer of the bone marrow and blood that is characterized by the swift increase of immature B- and T- lymphocytes. It is the most widespread pediatric cancer and the prime cause of cancer death among children. ALL is curable in &#62;85% of affected children, but adults and infants have considerably lower cure rates, and relapsed/refractory ALL has a 5-year survival of &#60;10%12. Thus, developing new treatment strategies to prevent or stop relapsed or refractory ALL is urgently needed. Although a number of xenograft models, such as mouse and rat13, have been developed, these methods are time-consuming (e.g., months) and expensive, and require comprehensive ethical regulations. To address an unmet clinical need for novel ALL therapy, developing a reproducible, time- and cost-effective animal model suitable for extending basic in vitro work is imperative.
In this study, we report the successful establishment of an in ovo xenografting protocol that reliably produces 3D vascular colonization of patient B- and T-ALL cells, which is potentially used for the design of novel therapy that could stop ALL progression and for the development of a predictive tool for ALL treatment outcome and risks.

<table><tbody><tr><td>34 G needles</td><td>BD Perisafe</td><td></td><td></td></tr><tr><td>Attune NxT Flow Cytometer</td><td>Thermo Fisher</td><td></td><td></td></tr><tr><td>BD Perisafe Syringe (32 G)</td><td>BD Perisafe</td><td></td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Millipore Sigma</td><td>A9647</td><td></td></tr><tr><td>Cell freezing medium</td><td>Gibco, ThermoFisher Scientific</td><td>12648010</td><td></td></tr><tr><td>Centrifuge (Allegra X-22R Centrifuge )</td><td>Beckman Coulter&amp;nbsp;</td><td></td><td></td></tr><tr><td>Disposable gloves</td><td>Royal Touch300 Nitrile Exam Gloves</td><td></td><td></td></tr><tr><td>Dissecting Microscope</td><td>Leica MZ16FA</td><td></td><td></td></tr><tr><td>DMEM</td><td>Gibco, ThermoFisher Scientific</td><td>11965092</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen, ThermoFisher Scientific</td><td>D12345</td><td></td></tr><tr><td>Egg incubator, Vevitts 120 rolling Incubator</td><td>Vixest Trading Co.</td><td></td><td></td></tr><tr><td>Ethyl alcohol (95%)</td><td>VWR International</td><td>BDH1158-4LP</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco, ThermoFisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Ficoll solution (Ficoll?? Paque Plus)</td><td>MilliporeSigma</td><td>GE17-1440-02</td><td></td></tr><tr><td>FITC-CD10 antibody</td><td>Santa Cruz Biotech</td><td>sc-46656 FITC</td><td></td></tr><tr><td>FITC-CD4 antibody</td><td>Santa Cruz Biotech</td><td>sc-19641 FITC</td><td></td></tr><tr><td>Fluorescence Microscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Hand drill (Dremel3000)</td><td>Bosch</td><td></td><td></td></tr><tr><td>HEK293T&amp;nbsp;</td><td>ATCC, VA, USA</td><td>CRL-3216</td><td></td></tr><tr><td>Hemocytometer</td><td>VWR international, Hausser Scientific</td><td>15170-263</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Millipore Sigma</td><td>H3149</td><td></td></tr><tr><td>Heparinized test tubes</td><td>BD Vacutainer&amp;nbsp;</td><td>BD 366480</td><td></td></tr><tr><td>Lipofectamine 3000</td><td>Invitrogen, ThermoFisher Scientific</td><td>L3000008</td><td></td></tr><tr><td>Multiplex Lentivirus Titer Kit (qPCR Lentivirus Titer Kit)</td><td>Abm</td><td>LV900</td><td></td></tr><tr><td>NH4Cl (Ammonium chloride_</td><td>Millipore Sigma</td><td>A9434</td><td></td></tr><tr><td>Olympus IX71 inverted microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>PE-CD19 antibody</td><td>Santa Cruz Biotech</td><td>sc-19650 PE</td><td></td></tr><tr><td>PE-CD8 antibody</td><td>Santa Cruz Biotech</td><td>sc-1177 PE</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)&amp;nbsp;</td><td>Gibco, ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P3813</td><td></td></tr><tr><td>Pipettes (adjustable volume)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Puromycin</td><td>Gibco, ThermoFisher Scientific</td><td>A1113803</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco, ThermoFisher Scientific</td><td>11875093</td><td></td></tr><tr><td>Scotch Cellotape</td><td>3M</td><td></td><td></td></tr><tr><td>Sterile 6-well plates</td><td>VWR international, Corning</td><td>22250-140</td><td></td></tr><tr><td>Sterile centrifuge tubes</td><td>Axygen Inc, Corning</td><td>AXYMCT-150-C-S</td><td></td></tr><tr><td>Sterile culture flasks (75T)</td><td>VWR International</td><td>10062-872</td><td></td></tr><tr><td>Sterile Falcon tubes (15 mL)</td><td>SARSTEDT</td><td>62.554.502</td><td></td></tr><tr><td>Sterile Falcon tubes (50 mL)</td><td>SARSTEDT</td><td>62.547.254</td><td></td></tr><tr><td>Sterile filter (0.45 &amp;micro;m filter)</td><td>VWR International</td><td>76778-974</td><td></td></tr><tr><td>Sterile forceps</td><td>VWR International</td><td>82027-404</td><td></td></tr><tr><td>Sterile glass slides</td><td>VWR international</td><td>48300-031</td><td></td></tr><tr><td>Sterile pipette tips</td><td>VWR International</td><td>76322-136, 76322-134, 76322-154</td><td></td></tr><tr><td>Sterile syringes</td><td>BD syringe</td><td>309628?</td><td></td></tr><tr><td>Trypan Blue</td><td>Invitrogen, ThermoFisher Scientific</td><td>T10282</td><td></td></tr></tbody></table>

in ovo xenografting of patient-derived acute lymphoblastic leukemia (all) cells (pdx-all)

In Ovo Xenografting of Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells (PDX-ALL)

This study introduces a rapid, cost-effective, and efficient method for in ovo xenografting of patient-derived acute lymphoblastic leukemia (ALL) cells, encompassing both B-cell and T-cell lineages. Using fertilized chicken embryos, we injected patient-derived B-ALL and T-ALL cells into the vasculature of embryos 11 days post-fertilization (11dpf). Remarkably, four days following the injection, the engrafted human leukemia cells exhibited significant survival, proliferation, and vascular colonization within the developing chicken embryo. By 15dpf, we detected a notable increase in CD10/CD19+ B-ALL and CD4/CD8+ T-ALL cells in blood samples from the embryo&#39;s vasculature, confirming successful engraftment. This system facilitates efficient and reproducible assessment of leukemia cell behavior in a living organism without the considerable costs and ethical constraints associated with other animal models. This rapid approach provides a high-throughput and biologically relevant in vivo platform for evaluating potential therapeutic compounds. The in ovo patient-derived xenograft (PDX)-ALL system presented here offers a promising tool for preclinical drug screening, mechanistic studies, and potentially for personalized medicine approaches in leukemia research.

To determine whether the engrafting of human ALL cells in developing chicken embryos was successful, 2nd-generation replication-incompetent lentivirus carrying mCherry were amplified and used them to label B- and T-ALL cell lines, SEM and MOLT316,17, respectively. As shown in Figure 1A, ~50% of B-(upper left two panels) and T-(upper right two panels) ALL cells were labeled with mCherry. Subsequently, patient-derived B-(#1 ...

This protocol describes in ovo&#160;xenografting of patient-derived B- and T-acute lymphoblastic leukemia (ALL) cells, which occurs 4 days following injection.

This study introduces a rapid, cost-effective, and efficient method for in ovo xenografting of patient-derived acute lymphoblastic leukemia (ALL) cells, ...

The scope of this research is cancer biology, with a focus on identifying therapeutic strategies to treat cancer. The in ovo patient-derived xenograft acute lymphoblastic leukemia model is constrained by a relatively short experimental window of around 10 days, limiting long-term studies on acute lymphoblastic leukemia progression or drug response beyond this timeframe. This protocol establishes a rapid cost-effective method for in ovo xenografting of patient-derived acute lymphoblastic leukemia cells, including B-cell and T-cell lineages.This protocol represents a promising tool for preclinical drug screening, mechanistic studies, and potentially personalized medicine approaches in leukemia research. The feasibility of using the established in ovo xenograft model for preclinical applications will be explored. To begin, transfer the procured eggs into a humidified rolling incubator set at approximately 50 to 60%humidity and a temperature of 39 degrees Celsius.Incubate the eggs in this incubator for 10 days post fertilization. Transfer the blood samples into a sterile 50-milliliter centrifuge tube and dilute each sample three times using two volumes of PBS. After centrifuging the sample along with a density gradient medium, collect the buffy coat layer of mononuclear cells from the interface and transfer it to a new sterile 15-milliliter tube.Add 10 milliliters of PBS to the tube and centrifuge the tubes at 300 G for five minutes to remove remaining serum components. Resuspend the pelleted cells in RPMI 1640 medium supplemented with 10%FBS. Use a hemocytometer to count the number of cells.Resuspend the cells in freezing medium consisting of 10%DMSO in FBS. Assess cell viability using the trypan blue exclusion assay and freeze the cells at minus 80 degrees Celsius and store them in liquid nitrogen. Next co-transfect 1.5 million HEK 293 T-cells with the desired plasmid vector using Lipofectamine 3000 and incubate the transfected cells for two days.Harvest the supernatant containing lentivirus from the wells and filter it through a 0.45-micrometer filter to remove debris. Transfer the filtered viral supernatant into centrifuge tubes and centrifuge at 20, 000 G for two hours at four degrees Celsius. For labeling, plate 1 million cells per milliliter of B-cell precursor SEM and T-cell MOLT3 cell lines as well as patient-derived B or T acute lymphoblastic leukemia cells into six-well plates containing RPMI 1640 with 10%FBS.Visualize mCherry-labeled cells under a fluorescence microscope. On day 10, examine the vasculature of the chick embryos under light to assess embryo viability, Gently wash the surface of each egg with 70%ethanol. Using a hand drill, drill into the air cell of each egg to create a small window approximately two centimeters in diameter.Seal the created window with transparent adhesive tape and return the eggs to a 5%carbon dioxide incubator. On day 11, inject 10 million mCherry-labeled acute lymphoblastic leukemia cells into the blood vessels of the developing embryos using 34-gauge needles. After sealing the window, return the eggs to a 5%carbon dioxide incubator and monitor embryo viability daily.On day 15, dissect the blood vessels out from the embryos. Place them on glass slides and photograph successful xenografts using a dissecting microscope equipped with fluorescence capabilities. Collect blood from the vasculature of chicken embryos using a sterile syringe with a 32-gauge needle.Transfer the blood into a 1.5-milliliter sterile tube containing heparin and centrifuge at 226 G for 10 minutes at four degrees Celsius. Label B-cell acute lymphoblastic leukemia cells with human FITC CD10 and human PE CD19 antibodies and T-cell acute lymphoblastic leukemia cells with human FITC CD4 and human PE CD8 antibodies at four degrees Celsius in the dark for 30 minutes. Wash the labeled cells twice using PBS containing 1%BSA and analyze using flow cytometry Vascular colonization by SEM and MOLT3 cell lines was clearly visible four days post injection, confirming successful engraftment in the developing chicken embryo vasculature.Patient-derived B-all and T-all cells showed strong fluorescent signals in the blood vessels at four days post-injection, indicating active colonization and proliferation in the vasculature. Flow cytometry revealed a significant increase in CD10/CD19 positive cells in embryos injected with SEM and patient-derived B-all cells at four days post injection compared to controls collected six hours post injection. Similarly, CD4/CD8 positive cells were significantly higher in embryos injected with MOLT3 and patient-derived T-all cells at four days post injection.

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Research

JoVE Journal

Cancer Research

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