Name: Murine Model of Leukemia Relapse to Induction Chemotherapy for Acute Lymphoblastic Leukemia (Video) | JoVE Journal
Uploaded: 2025-10-17T13:00:00.000+00:00
Duration: 8 min 31 s
Description: Learn how to establish a B-ALL PDX relapse model in mice to investigate drug resistance mechanisms in acute lymphoblastic leukemia. This method enables dynamic monitoring of resistance evolution beyond minimal residual disease.

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5 minutes

We thank the Centro Infantil Boldrini and all the patients who donated their samples for the study. Manuella M. Hoff received a fellowship by the Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES) from the S&#227;o Paulo Research Foundation (FAPESP, 2024/ 03383-2). Jos&#233; A. Yunes received a productivity fellowship from the National Counsel of Technological and Scientific Development (CNPq, 308399/2021-8). This work was supported by research funding from the Brazilian Ministry of Health by the PRONON program (Programa Nacional de Apoio &#224; Aten&#231;&#227;o Oncol&#243;gica, NUP 25000.211174/2019-45).

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<li>Alruwetei, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Examining+treatment+responses+of+diagnostic+marrow+in+murine+xenografts+to+predict+relapse+in+children+with+acute+lymphoblastic+leukaemia%5BTitle%5D)+AND+%22Br+J+Cancer%22%5BJournal%5D)">Examining treatment responses of diagnostic marrow in murine xenografts to predict relapse in children with acute lymphoblastic leukaemia.</a> Br J Cancer. 123 (5), 742-751 (2020).</li>
<li>Wunderlich, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+chemotherapy+modeling+with+RAG-based+immune+deficient+mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Improved chemotherapy modeling with RAG-based immune deficient mice.</a> PLoS One. 14 (11), e0225532(2019).</li>
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The authors declare no conflicts of interest.

Understanding the biological mechanisms underlying neoplastic diseases is essential for the development of more effective therapies and for improving patient outcomes. In conditions such as pediatric acute lymphoblastic leukemia, these investigative approaches are not only relevant for increasing cure rates but also hold particular value for patients in whom standard treatments have reached the limits of therapeutic intensity and tolerability. In the setting of relapse and treatment resistance, the challenge can no longe...

Acute lymphoblastic leukemia (ALL) is the most prevalent cancer in children. Despite the high cure rates achieved in recent decades with 5-year overall survival reaching 90% in high income countries1 and major efforts in the search for new therapeutic strategies, in developed countries, 15%-20% of patients relapse from the disease, to who the survival rate varies between 30%-60% depending if the first relapse occurs early (under 18 months) or later (36 months) after diagnoses2,3. In view of this, refractory and relapse ALL remain challenges to be overcome. In terms of diagnosis, refractory ALL is distinguished from relapse by the poor sensitivity to chemotherapy, while relapse ALL is characterized by the recurrence of the disease after a period of remission. As with other cancers, ALL is characterized by genetic abnormalities, including aneuploidies, chromosomal rearrangements, and various gene mutations that are associated with different therapeutic responses and relapse risk4. Since ALL relapses occur more frequently in high-risk patients - who have already received maximum-intensity chemotherapy - further adjustments to drug doses or combinations are unlikely to be sufficient. Instead, efforts must be directed toward identifying the mechanisms underlying drug resistance and relapse.
In light of this, researchers have attempted to understand how the selection pressure exerted by chemotherapy contributes to the emergence of resistant cells. Most of the existing literature attributes relapse to the propagation of a resistant malignant subclone that was already present at the time of diagnosis5. However, a recent study suggests the existence of alternative mechanisms, such as epigenetic modifications that regulate transcriptional programs, which may also contribute to relapse6.
Substantial effort has been devoted to creating accurate in vivo models to study ALL relapses. Immunodeficient mice have been successfully used for ALL engraftment, preserving the original disease characteristics7,8, leading to the development of patient-derived xenografts (PDXs). This model allows for the engraftment and proliferation of original tumor/leukemia cells in a compatible microenvironment. One of its major advantages is the ability to assess the response of transplanted ALL cells to chemotherapy9.
PDX models have also been employed to mimic and study ALL relapses. For example, a protocol developed by Samuels et al.10 used non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice transplanted with patient leukemia cells to investigate resistance mechanisms to an induction-like chemotherapy regimen. The authors described treatment with vincristine, dexamethasone, L-asparaginase, and daunorubicin (VXLD) and provided examples of the number of treatment cycles and timing of relapse. However, the model focused on analyzing cells immediately after treatment - those that survived initial therapy - likely representing minimal residual disease (MRD) rather than relapse.
A more recent study evaluated whether short-term treatment of ALL-transplanted NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NOD scid gamma, or NSG) immunodeficient mice could predict relapse in corresponding patients11. Intermediate-risk ALL samples - from both relapsing and non-relapsing patients - were transplanted into mice and treated with an induction-like regimen consisting of vincristine, dexamethasone, and L-asparaginase (VXL). Time to engraftment and post-treatment relapses were measured and compared. The results suggested that this PDX model could serve as a prognostic tool for predicting patient outcomes. This finding supports the utility of PDX mice models in studying the biology of ALL relapses. For instance, relapsing cells could have been examined at different time points to uncover the mechanisms underlying relapse.
In this manuscript, we present improvements to the PDX mice model for studying B-ALL relapse. We aim to study disease recurrence and the characteristics of relapsed cells after their shaping by induction chemotherapy. A four-drug, induction-like regimen - vincristine, dexamethasone, L-asparaginase, and daunorubicin - was standardized, using the NSG mice strain. This strain is known to have limitations in withstanding repeated chemotherapy cycles due to the Prkdcscid mutation and increased sensitivity to genotoxic stress12. Therefore, animal body weight was monitored as a marker of treatment toxicity.The B-ALL relapse model can be divided into three main steps: (1) transplantation of ALL cells into mice, aiming for an engraftment threshold of 0.2-1% human CD45&#8314; cells in peripheral blood; (2) 4 weeks of chemotherapy treatment, followed by a resting period until the first relapse (R1); and (3) upon detection of R1, repeated 1 week induction-type treatment cycles. Subsequent relapses (R2, R3...RN) may occur, and the interval between treatment and relapse (&#916;t) is recorded. A progressive shortening of &#916;t is interpreted as an indication of acquired drug resistance.As part of more in-depth investigations into the mechanisms of relapse, B-ALL cells may be collected before and after different stages of the protocol. It is important to emphasize that, depending on the time between relapse occurrence and cell recuperation, the inherent mechanisms of this phenomenon may not be conserved. In view of this, it is extremely important that relapse cells are collected rapidly after relapse confirmation. The cells collected can be used for future phenotypic and genetic analyses, including assessments of cell cycle dynamics, cell migration, leukemia-initiating cell frequency, chromosomal alterations, and epigenetic modifications, among others.
For the purposes of this article, we will focus on describing the relapse mouse model. Detailed information will be provided for the three experimental phases - transplantation, treatment until first relapse (R1), and cyclic treatment until multiple relapses (R2, R3...). We hope this model will allow researchers to generate chemotherapy-resistant B-ALL cells from diverse subtypes, enabling deeper investigation into the cellular and molecular changes associated with relapse. Ultimately, this may contribute to a better understanding of resistance mechanisms and facilitate the development of more effective and personalized therapies.

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murine model of leukemia relapse to induction chemotherapy for acute lymphoblastic leukemia

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

Acute lymphoblastic leukemia (ALL) is a neoplasm of immature lymphoid precursor cells and one of the most common childhood malignancies. Although cure rates in pediatric ALL approach 90%, relapse and drug resistance remain challenges. In this context, studying cancer biology in vivo is essential, and patient-derived xenografts (PDX) using murine models are widely used for this purpose. However, PDXs from relapsed samples - or compared to diagnostic ones - do not fully assess the mechanisms behind relapse initiation and progression. This protocol relies on generating B-ALL PDX relapses in mice, allowing collection of leukemic cells throughout the process of acquiring drug resistance. Animals engrafted with B-ALL PDX cells are treated with a combination of four drugs commonly used in ALL therapy, representing distinct mechanisms of action: dexamethasone, vincristine, L-asparaginase, and daunorubicin. After a 4-week remission induction phase, animals are monitored for relapses during a resting period. The cycle of treatment and relapse is repeated until the time to relapse shortens, serving as a surrogate for increased in vivo drug resistance. To explore mechanisms underlying relapse and resistance, leukemic cells are collected before and after each treatment cycle for phenotypic and genetic analysis. To validate the model, different pediatric ALL samples were tested, including both good and poor responders, as assessed by the presence of blasts in peripheral blood after 7 days of corticosteroid therapy and by minimal residual disease (MRD) at days 15, 33, and 78/96. The results observed in mice were consistent with patient data, confirming the model&#39;s accuracy in mimicking the clinical response to chemotherapy. This approach enables the dynamic study of relapse and drug resistance in B-ALL, supporting mechanistic investigations and the development of novel therapeutic strategies.

In the context of our studies, precursor B-cell ALL samples were selected from two patient groups: one who exhibited a favorable response, as determined by the absence of leukemic cells in day 8 peripheral blood and absence of residual leukemia cells in bone marrow at days 15, 33, and 78/96. The other group was formed by ALLs that matched the molecular subgroups of the previous ones, obtained from patients who had a poor clinical response as evaluated by Minimal Residual Disease (MRD), as...

The protocol aims to describe a mouse model for generating relapses of acute lymphoblastic leukemia, based on the dynamics of the response to induction chemotherapy.

Acute lymphoblastic leukemia (ALL) is a neoplasm of immature lymphoid precursor cells and one of the most common childhood malignancies. Although cure ...

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.

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Research

JoVE Journal

Cancer Research

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

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.

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

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Modeling Chemotherapy Resistant Leukemia In Vitro

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-Grünwald Giemsa staining.

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

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

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.

Assessment of Chimeric Antigen Receptor T Cell-Associated Toxicities Using an Acute Lymphoblastic Leukemia Patient-Derived Xenograft Mouse Model

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.

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in 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.

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

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

This protocol demonstrates temporal vein injection for intravenous delivery of pediatric cancer cells to neonatal mice, enabling precise, reproducible modeling of early-life immune responses against cancer. The method enables assessment of the impact of interventions, such as immunomodulation or therapy, when applied during the neonatal period.

A Neonatal Intravenous Transplant Model for Assessment of Early-Life Immune Activities Against Cancer Cells

Learn how to establish a B-ALL PDX relapse model in mice to investigate drug resistance mechanisms in acute lymphoblastic leukemia. This method enables dynamic monitoring of resistance evolution beyond minimal residual disease.

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

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