Murine Model of Leukemia Relapse to Induction Chemotherapy for Acute Lymphoblastic Leukemia

Our research establishes a B-ALL PDX relapse model in mice to investigate how leukemic cells acquire drug resistance. We hope to uncover mechanisms driving relapse that may guide future therapeutic strategies. Our main challenge is the lack of an immune system in the NOD/SCID mice, limiting leukemia-immune interactions.

And a potential solution is adapting the protocol to remove leukemia in immunocompetent hosts. Our study addresses gaps by naming exploration of chemotherapy-resistant cells while under treatment in vivo, capturing dynamic resistance evolution beyond MRD. Our protocol generates human pediatric AML-resistant cells in vivo using PDX models to mimic full clinical relapse cycles, enabling access to relapse biology often unattainable through in-vitro systems.

Our model paves the way to explore genetic and epigenetic drivers of relapse, microenvironmental influences on resistance, patient-specific viability, and to test novel therapies, including immunotherapies, against relapsed B-ALL. To begin thaw cryopreserved acute lymphoblastic leukemia cells that were obtained from the patient's bone marrow aspirate. Transfer the thawed sample into a 15-milliliter centrifuge tube and add 14 milliliters of sterile PBS to wash the cells off the cryopreservation solution.

Place the tube into a centrifuge and spin at 300g for five minutes. Then carefully remove and discard the supernatant without disturbing the pellet. Resuspend the cell pellet in sterile PBS to obtain around 5 to 10 million viable cells in 100 microliters for each animal.

Keep the prepared transplant solution on ice until transplantation. For transplantation, select two-month-old male or female NSG mice for leukemia transplantation. Place one mouse at a time in a secure and approved mouse restrainer, ensuring the tail remains outside and easily accessible for transplantation.

Now, position the restrained mouse 30 centimeters from an infrared light source to ensure proper vein expansion. Adjust the exposure time based on the power of the lamp, keeping the tail exposed to medium power light for five minutes. Next, using an insulin syringe with an attached 12.7 by 0.33 millimeter needle, inject 100 microliters of the transplant solution into the tail vein.

After injection, place the transplanted mouse into a specific pathogen-free facility and allow it to rest for 15 days. Retrieve the animal for blood collection and restrain it securely by holding its head. With the right hand puncture the facial vein of the submandibular plexus using a sterile, sharp disposable lancet.

Collect 50 microliters of peripheral blood from each mouse in a 1.5 milliliter centrifuge tube, containing eight microliters of 50-millimolar EDTA two weeks after inoculation. After mixing, pipette 50 microliters of the sample into a flow cytometry tube. Prepare an antibody mixture consisting of anti-mouse CD45 antibody, anti-human CD45 antibody, anti-human CD19 antibody, and sterile PBS as the diluent.

Add 10 microliters of the antibody mixture to each blood sample, and gently tap the tube with the index finger to homogenize the sample. Incubate the samples for 30 minutes at room temperature while shielding them from light. After washing the leukemia cells with PBS, resuspend them with 200 microliters of sterile PBS by gently tapping the cytometry tube with the index finger.

Employ standard flow cytometry methodologies to determine the percentage of human CD45 positive cells in the peripheral blood relative to mouse CD45 positive cells. Initiate treatment when the percentage of human CD45 positive cells in the peripheral blood reaches a median range between 0.2%and 1%Weigh the animals designated for treatment and calculate the arithmetic mean of the weights to determine the appropriate dose of each drug for the group. Now, manually restrain the animal with the left hand, keeping the ventral portion facing upward.

Locate the lower-right quadrant of the abdomen. Using a sterile, sharp, single-use injection needle, inject 100 microliters of the drug solution into the lower-right quadrant intraperitoneally. To recover the leukemic cells use sterile surgical scissors and tweezers to make a small incision in the animal's abdominal skin and remove it.

Make a small incision in the peritoneum, and extend the incision until there is full access to the abdominal organs. Locate the spleen beneath the stomach and remove it carefully using tweezers. Place the extracted spleen in a container with sterile PBS for further processing.

For femur harvesting, open the peritoneal cavity to gain access to the hind legs of the animal, and locate the femur bone. Using scissors, cut at the joints that connect the femur with the pelvis and with the tibia and fibula to remove it. Place the harvested cells in a 15-milliliter centrifuge tube and spin at 420g for 30 minutes using a swing-bucket rotor without acceleration and without brake to preserve the separation of phases.

Using a sterile disposable Pasteur pipette carefully remove and discard the top aqueous phase. With a new sterile, disposable Pasteur pipette, collect the leukemic cell layer that has formed into a fresh 15-milliliter centrifuge tube. Finally, cryopreserve the isolated leukemic cells following standard cell culture cryopreservation guidelines.

In the mouse model transplanted with leukemic cells from good-responder patients, VXLD treatment induced remission. But with repeated cycles, the interval between remission and relapse became progressively shorter, indicating drug resistance. In the mouse model transplanted with leukemic cells from poor-responder patients, VXLD treatment failed to induce remission and only slowed disease progression for three weeks before leukemic load exceeded tolerable limits, demonstrating refractoriness.

In another poorly responding leukemia, VXLD treatment temporarily induced remission, but relapse occurred rapidly after a single rest week without treatment, indicating short-lived remission.