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

Summary

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

Abstract

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'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.

Introduction

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 cells^{2,3,4,5,6,7,8,9,10}, 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 αVβ3, 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 >85% of affected children, but adults and infants have considerably lower cure rates, and relapsed/refractory ALL has a 5-year survival of <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.

Protocol

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

1. Procure fertilized chicken eggs. Obtain ~10% more eggs than needed to consider the number of eggs damaged during shipping or the cracking procedure.
2. Store the fertilized eggs for up to two weeks at 12 °C without losing viability.
3. Transferthe eggs into a humidified (~50%-60%) rolling incubator at 39 °C.
4. Incubate the eggs in the incubator for 10dpf. Note that day 1 starts when the eggs are transferred into the incubator.

2. Isolation of acute lymphoblastic leukemia cells from blood samples from patients with ALL

1. Isolate the cells as described previously^14,15.
2. Collect 10 mL of peripheral blood (or marrow) from B- and T-ALL patients into heparinized test tubes.
3. 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.
4. 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.
5. 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.
6. 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).
7. 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).
8. Wash the cells in 1x PBS as described in step 2.6.
9. Resuspend the cells in RPMI 1640 containing 10% FBS and count the number of cells using a hemocytometer.
10. Assess cell viability using the Trypan Blue Exclusion assay.
11. Resuspend the cells in the freezing medium (10% DMSO in FBS), freeze them at -80 °C, and store them in liquid nitrogen.

3. Amplification of 2^nd-generation replication-incompetent lentivirus carrying mCherry

1. 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 °C in a humidified incubator with 5% CO₂.
2. Cotransfect the cells (1.5 x 10⁶) with FUW-Luc-mCherry-puro transfer, pCMVΔr8.91 packaging and pVSV-G envelope vectors at a ratio of 3:2:1 using Lipofectamine 3000 for 2 days.
3. Harvest the lentivirus-containing supernatant and filter through a 0.45 µm filter.
4. Transfer the cleared viral supernatant into centrifuge tubes, centrifuge at 20,000 x g for 2 h at 4 °C, discard the supernatant, and resuspend the pellet in 100 µL of PBS or serum-free medium.
5. Measure the multiplicity of infection (MOI) using the qPCR Lentivirus Titer kit.

4. Labeling of continuously growing ALL cell lines and patient-derived ALL cells with mCherry

1. 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.
2. Infect the cells with 300 µL 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 °C with 5% CO₂ for 2 days.
3. Add 1 µg/mL puromycin to the culture medium to select stably transduced cells for 2 days.
4. Visualize mCherry-labeled cells with a fluorescence microscope.

5. In ovo xeno grafting

1. 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.
2. Drillinto the air cell of the eggs to create a small (~2.0 cm in diameter) window using a hand drill.
3. Seal with transparent adhesive tape and return to a 5% CO₂ incubator.
4. 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.
5. Seal the windows with transparent adhesive tape, return to 5% CO₂ incubator, and monitor the embryo viability daily.
6. 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.

6. Flow cytometry

1. 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 °C.
2. Remove the supernatant (plasma) and resuspend the cell pellet in 1x PBS containing 1% BSA (bovine serum albumin).
3. Perform step 2.7 and step 2.8 to lyse red blood cells.
4. 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 °C in the dark for 30 min.
5. Wash the labeled cells twice with PBS containing 1% BSA, centrifuge at 226 x g for 5 min at 4 °C, resuspend in 1x PBS, and analyze by flow cytometry.
6. 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 vs. SSC): use FSC-A 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 vs. FSC-A): perform doublet discrimination by gating on FSC-H vs. FSC-A, ensuring only single-cell events are analyzed¹⁸.
7. 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 ± SEM of three independent experiments (n = 3).

Results

To determine whether the engrafting of human ALL cells in developing chicken embryos was successful, 2^nd-generation replication-incompetent lentivirus carrying mCherry were amplified and used them to label B- and T-ALL cell lines, SEM and MOLT3^16,17, 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 ...

Discussion

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 µL) to ensure proper vascular delive...

Materials

List of materials used in this article

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