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

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.