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

Summary

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.

Abstract

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'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.

Introduction

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 diagnoses^2,3. 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 characteristics^7,8, 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-Prkdc^scidIl2rg^tm1Wjl/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 Prkdc^scid 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⁺ 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 (Δt) is recorded. A progressive shortening of Δt 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.

Protocol

All animal experiments were performed according to the regulations and ethical guidelines of Comissão de Ética no Uso de Animais from Centro Infantil Boldrini (CEUA/Boldrini 0047-2024). The leukemia samples used in this study were obtained from bio-banked vials of patients treated at Centro Infantil Boldrini, who allowed the use of their cells through an informed consent form. The project was approved by the Institutional Ethics Committee (CAAE 34601120.7.0000.5376).

1. Leukemia cell transplantation and engraftment monitoring

1. Preparation of leukemia samples for mouse transplantation
   1. Thaw previously cryopreserved samples of ALL cells taken from the patient's bone marrow aspirate. Use a water bath set at 37°C. Thaw the samples by moving the cryotube in a circular motion.
   2. Transfer the thawed sample to a 15 mL centrifuge tube. Add 14 mL of 1x sterile PBS to the sample to wash the cells and remove the cryopreservation solution. Centrifuge the tube at 300 x g for 5 min. Remove the supernatant.
2. Preparation of transplant solution
   1. Resuspend the thawed cells in sterile 1x PBS. Use 5-10 x 10⁶ viable cells in a volume of 100 µL of PBS for each animal to be transplanted. Keep the Transplant Solution on ice until transplantation.
      NOTE: Cells were counted following the semi-automatic method¹³.
3. Mice transplantation
   1. Select 2-month-old male or female NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice for leukemia transplantation.
      NOTE: To maintain a homogeneous experimental design free of animal sex interference, the ALL samples were transplanted into both mouse sexes, ensuring that all the good and poor responder leukemias were equally represented in both sexes. As a result, we found that female mice engraft earlier than male mice. Considering that, to avoid possible interference of mouse sex in future analyses (such as engraftment kinetics), we suggest using only one mouse sex when working with the same leukemia sample. No other parameters were used to preferentially choose one sex over the other.
   2. Place one mouse at a time in a secure and approved mouse trap to ensure proper containment of the animal. Ensure that the animal's tail is outside the trap and easily accessible for transplantation.
      NOTE: Leukemia cells are transplanted into mice by intravenous (i.v.) injection. Established protocols use the lateral veins of the tail for this purpose¹¹.
   3. Use infrared light to ensure proper vein expansion. Inject 100 µL of the Transplant Solution using a sterile, sharp, single-use injection needle. Keep the transplanted mice in a specific pathogen-free facility. Allow them to rest for 15 days.
      1. To use the infrared light, keep the already restrained animal outside the cage and 30 cm distant from the light. Depending on the potential light power, adjust the time of exposure. When using a medium power light, a period of 5 min is sufficient to ensure vein expansion. Leaving the tail exposed for a longer period may cause burns and stress to the animal.
      2. For the injection, use an insulin syringe with an attached needle, with a volume of 1 mL and a needle size of 12.7 mm x 0.33 mm. The specification of the needle used can be found in the Table of Materials. Maintain mice in a 12 h/12 h light/dark cycle regimen. In addition, house the animals used in this study in an autoclavable translucent amber polycarbonate cage with environmental enrichment to create nests.
4. Peripheral blood collection
   1. Collect a 50 µL peripheral blood sample from each mouse 2 weeks after inoculation and every week thereafter.
   2. Manually restrain the animal with the left hand, ensuring that its head is held securely. With the right hand, use a sterile, sharp, disposable lancet to puncture the facial vein of the animal's submandibular plexus.
      NOTE: This is a potentially harmful procedure for the mice and must be done carefully.
      1. Make sure the animal can breathe easily during the restraint. Watch for changes in respiratory rate (shortness of breath) and changes in mouth color (it should not turn purple or dark blue). If the submandibular plexus is not punctured correctly, bleeding in the ear may occur because of the ear canal puncture. To guide future experimenters, Supplementary Figure 1 is available to ensure the proper location of the plexus.
         NOTE: As an alternative, tail vein bleeds can also be used to collect blood samples.
   3. After the collection, do not leave the animal unattended. It must be active and unharmed. If the mouse shows signs of falling (head or body) and inactivity, it should be euthanized. Do not exceed 50 µL volume, as larger amounts of blood may result in death of the animal.
   4. Keep the collected blood sample in a 1.5 mL centrifuge tube containing 8 µL of 50 mM EDTA to avoid sample coagulation.
      NOTE: Here, we used a previously prepared EDTA solution as an anticoagulant. If preferred, commercially available low-volume EDTA tubes for blood collection can also be used at this step. The blood samples must be processed on the same day.
5. Preparation of peripheral blood samples for processing
   1. Homogenize the blood with EDTA. Transfer 50 µL of the mixture into a flow cytometry tube.
   2. Prepare an antibody mixture to be added to each sample consisting of anti-mouse CD45 antibody (mCD45), anti-human CD45 antibody (hCD45), anti-human CD19 antibody (hCD19) and sterile 1x PBS as diluent.
6. Blood samples processing
   1. Add 10 µL of the antibody mixture to each blood sample. Homogenize by gently tapping with the index finger. Incubate for 30 min at room temperature, shielded from light.
      NOTE: Select the appropriate antibodies described above, considering the available channels of the cytometer. To determine the amount of antibody used per sample, consider performing a titration curve for each antibody. The antibodies used in our study, the fluorochromes, and the volume chosen for the samples are listed in Supplementary Table 1. A complete description of the antibodies catalogue numbers and clone names is listed in the Table of Materials.
   2. Add 1 mL of commercial 1x Red Blood Cell (RBC) Lysis Solution to each sample. Homogenize vigorously with index finger. Incubate for 15 min at room temperature, shielded from light.
      ​NOTE: The lysis solution used in this step is a commercially available 10x RBC Lysis Solution. If a different reagent is used, the 1x working volume for lysis must be adjusted.
   3. Add 2 mL of sterile 1x PBS to each sample for the first wash step. Centrifuge samples at 200 x g (in a swing rotor) for 5 min at room temperature. Gently discard the supernatant by inverting and dry any remaining liquid at the top of the tube by gently blotting with paper towels.
      NOTE: In the washing steps, the leukemia cells will be pelleted at the bottom of the cytometer tube, and the remaining fluid will be discarded. It is imperative to exercise caution when handling the samples, as in certain instances, the pellet may not be discernible. To ensure the integrity of the pellet, avoid shaking the sample, as this can result in its loss.
   4. Repeat step 1.6.3. Resuspend the leukemia cells with 200 µL of sterile 1x PBS by gently tapping the cytometry tube with index finger. Samples are ready to be analyzed by flow cytometry. Engraftment monitoring must be performed on the same day as blood collection.
7. Engraftment monitoring by flow cytometry
   1. Employ standard flow cytometry methodologies^9,14 to determine the percentage of human CD45+ cells in the peripheral blood relative to mice CD45+ cells (% hCD45+).
   2. Collect data generated by flow cytometry for subsequent analysis of the percentage of hCD45+ over total CD45 cells (hCD45+ plus mCD45+) using flow cytometry analysis software.
      NOTE: To ensure proper monitoring of engraftment, the following analysis steps should be performed on the same day. This daily sequence is particularly important when considering the start of the future treatment schedule, which we suggest beginning on Monday. In the impossibility of beginning on Monday, the treatment can begin on any day of the week, depending on the achievement of the hCD45+% (0.2%-1%) on peripheral blood.
8. Preparation of the environment for flow cytometry data analysis
   1. Launch flow cytometry analysis software. Import the data collected and wait for its recognition by the tool.
   2. Open the data from the initial sample to create the gate labeled Lymphocytes. Verify axis settings: select FSC-A for the X-axis and SSC-A for the Y-axis. In the upper gate creation panel, select the poly-liner tool and outline the main cell population.
      NOTE: Lymphocytes typically appear between 50,000-100,000 on the X-axis and 0-50,000 on the Y-axis. Values may vary depending on the characteristics of each leukemia.
   3. Double-click the Gated Population to create a new gate. To exclude cell doublets, set FSC-A on the X-axis and FSC-H on the Y-axis. Use the poly-liner tool to isolate the primary cell population. Draw the gate closely around the population, excluding events distant along the X-axis.
      NOTE: This step distinguishes individual cell events.
9. Creating the cytometry panel to distinguish cell populations
   1. Double-click the Selected Population to differentiate human from mouse CD45 cells. Set the X-axis to the mCD45 fluorophore and the Y-axis to SSC-A. Using the shape tool, draw a square around the mCD45-negative population.
   2. Double-click the Resulting Population to distinguish hCD19+ and/or hCD45+ cells. Set the X-axis to the hCD19 fluorophore and the Y-axis to hCD45. In the gate creation panel, select Perpendicular Lines to separate the distinct populations.
      NOTE: As an alternative, the investigator can use the gate or possibility for discriminating hCD45 and mCD45 cells. This possibility considers the sum of events as 100%, creating a more precise strategy for identifying populations.
10. Applying statistics to the created panel
    1. Calculate the percentage of each isolated cell population relative to all acquired events. In the main software window, right-click the Subgraph icon derived from each sample's main graph and select Create Statistics. In the opened window, click Frequency of and select Single Cells. Perform this step individually for each subgraph.
    2. Select all modifications made to apply the gating strategy and associated statistics to all samples. Then, using the left mouse button, drag the selected modifications to the All Samples option in the main software window.
    3. Click Table Editor in the main window to generate a table with all calculated frequencies. In the pop-up window, choose the .xls file format, designate the destination folder, and click Create Table.
       NOTE: All steps performed using the flow cytometry analysis software can be visualized in Supplementary Figure 2. The final table generated by this step contains the percentage of hCD45+ necessary to generate graphs to analyze the engraftment progression over time.
11. Plotting of engraftment progression
    1. Launch a graph plotting and analysis software. Select the XY Table Format. In the main window, choose Enter or Import Data into a New Table, set X to Numbers, and Y to Enter replicate values in side-by-side subcolumns.
    2. In the opened table, organize the column titles: enter Days of engraftment for the X column and % hCD45+ on PB for the first group of Y subcolumns. Complete the X column with the days post-injection corresponding to the days of blood collection for engraftment monitoring. Manually enter the weekly collected percentage of hCD45+ data for each mouse in the Y subcolumns.
    3. In the software menu, under the Family subset, select Data 1. In the pop-up window, choose the second graph option named Points & Connecting Line with Error Bars, and enable plotting of mean and error bars.
    4. Adjust the graph axes to the desired ranges. Click Export in the top right corner of the menu to download the final graph.
       NOTE: All data collected during the experiments with any single ALL must be plotted on the same graph to facilitate analyses over time since the start of the experiment. A descriptive example of graph plotting can be found in Supplementary Figure 3.

2. Treatment of mice

1. Preparation of drug working solutions
   1. Prepare a vincristine working solution at a concentration of 1 mg/mL. Prepare a dexamethasone working solution at 4 mg/mL. Prepare L-asparaginase working solution at 2,000 IU/mL. Prepare the daunorubicin working solution at 2 mg/mL.
      NOTE: The drugs utilized in this protocol are equivalent to those employed in the clinical treatment of patients¹⁵. Dexamethasone working solution vehicle is composed of creatinine, sodium citrate dihydrate, sodium hydroxide, sodium bisulfite, methylparaben, propylparaben, and water for injection. Vincristine working solution vehicle is composed of mannitol, sulfuric acid, sodium hydroxide, and water for injection. L-asparaginase and daunorubicin are frequently available in lyophilized form. In such cases, the recommended vehicle for reconstitution is sterile water for injections, which must be used to achieve the correct concentration. To prevent loss of drug activity due to repeated cycles of thawing, drugs must be divided into working aliquots used no more than 3x.
2. Animals weighing when leukemia is detected
   1. Initiate the treatment at the point at which the percentage of hCD45+ cells in the peripheral blood of mice reaches a median range between 0.2% and 1%. Upon reaching it, divide the transplanted mice into two distinct groups: one to receive treatment and one to serve as a control.
   2. Upon attaining the established threshold, persist in the weekly monitoring of leukemia progression. Continuously collect hCD45% data to monitor responsiveness to treatment, the occurrence of relapses, and the detection of drug resistance.
   3. Weigh the animals to be treated. Use the arithmetic mean of weights to ascertain the dose of each drug to be administered to the group. Weigh the control group animals to compare weight gain/loss between groups.
      1. Use the following drug doses: vincristine, 0.15 mg/kg; dexamethasone, 5 mg/kg; L-asparaginase, 1,000 IU/kg; and daunorubicin, 1.2 mg/kg. Weigh all animals at least 3x per week: on Monday, Wednesday, and Friday. Use the weights to adjust treatment doses of the drugs and to monitor the level of treatment toxicity.
3. Preparation of the drug administration solutions from the working solution aliquots
   NOTE: Drug administration solutions consist of sterile 1x PBS as diluent and the correct volume (according to the animal's weight) of working drug solutions (sterile water is used exclusively for reconstitution and not for injection) administered each day of the week. Drugs that are administered intraperitoneally can be united in the same solution; however, if any drug is administered intravenously, it should be placed in an individual solution. The volume of the drug administration solution to be administered to each animal is 100 µL.
   ​NOTE: A sterile insulin syringe with attached needle (1 mL, with needle dimensions 12.7 mm X 0.33 mm) was used in the step described above. A complete description of the syringe can be found in the Table of Materials.
   1. On Monday, prepare a drug administration solution consisting of dexamethasone, vincristine, and PBS. Administer the drugs intraperitoneally.
   2. On Tuesday and Thursday, prepare a drug administration solution consisting of dexamethasone, L-asparaginase, and PBS. Administer the drugs intraperitoneally.
   3. On Wednesday, prepare a drug administration solution consisting of dexamethasone and PBS and administer it intraperitoneally. Prepare a second drug administration solution consisting of daunorubicin and PBS. Administer it intravenously.
   4. On Friday, prepare a drug administration solution consisting of dexamethasone and PBS. Administer the drug intraperitoneally.
   5. Allow the animals to rest during the weekend days. Repeat the cycle of treatment for 4 weeks.
      NOTE: A comprehensive treatment schedule can be found in Supplementary Table 2. It is important to clarify that other protocols already established for mouse treatment were tested, but resulted in high levels of toxicity for the NSG strain. Therefore, the treatment schedule proposed was first submitted to a standardization process for the reduction of toxicity, which resulted in the doses and frequency of administration presented in this protocol. It is expected that the percentage of hCD45+ in the treated group will decrease compared to the percentage of hCD45+ in the control group due to treatment administration.
4. Drug administration and monitoring of leukemia responsiveness
   1. Manually restrain the animal with the left hand to administer drugs intraperitoneally. Keep the animal with the ventral portion up. On the ventral portion, locate the lower right quadrant.
   2. With the right hand, use a sterile, sharp, single-use injection needle and inject 100 µL of the drug administration solution into the mice's lower right quadrant.
      NOTE: Be careful when restraining the animal; it should not be a harmful procedure for the mice. As in blood collection, be aware of any respiratory complications. In the case of intraperitoneal injections, always hold the needle perpendicularly in order to avoid organ drilling.
   3. Follow the same instructions used to transplant leukemia cells to administer drugs intravenously. Allow mice to rest after 4 weeks of treatment administration until reappearance of ALL cells, here named first relapse (R1).
      NOTE: Despite the difference in the administration route, the intravenous injection uses the same volume as injected intraperitoneally. If any animal shows signs of suffering from a leukemic burden, such as facial expressions indicating pain, loss of appetite, sudden weight loss or decreased mobility, a humane early endpoint can be applied. Humane euthanasia follows the euthanasia procedure described in steps 4.4.1 to 4.4.2 further on.
5. Monitoring of leukemia responsiveness and detection of first relapse (R1)
   1. Do not discontinue weekly flow cytometry monitoring of leukemia during treatment or resting phases.
      NOTE: Considering the 5-day-a-week treatment, it is strongly recommended that monitoring be performed at the beginning of the week. In our study, blood collection, sample processing, and analysis were always performed on Mondays.
   2. Perform blood collection and any subsequent procedures required for monitoring according to steps 1.4 - 1.11.
      NOTE: After the given resting period, R1 cells will be detected by the sudden increase in the percentage of hCD45+ in the peripheral blood of mice. The period between the end of treatment and the occurrence of relapse is variable and can be distinct for each leukemia screened. At the detection of R1, the third and final phase of the proposed mouse model of relapse should be performed. To improve monitoring and response analyses, we suggest using objective response measures such as progressive disease, stable disease, level of responsiveness (partial, complete, or maintained), remission, and resistance, following the guidelines described by Randall et al. (2024)¹⁶.

3. Repeated cycles of treatment until emergence of resistant cells

1. Detection of first relapse
   1. Administer a full week of induction-type treatment to any mouse having leukemia relapse (named R1), i.e., when the threshold of 0.2% hCD45+ in peripheral blood is reached. For 1 week, from Monday-Friday, follow the drug administration schedule used for the 4-week induction-type treatment.
      NOTE: The administration of only 1 week of treatment at this point aims to test in vivo whether the leukemia cells have gained resistance to chemotherapy while eventually driving leukemia progression to successive relapses over time, under chemotherapy pressure. We chose not to administer a second 4-week schedule as a means to avoid excessive toxicity and loss of animals.
   2. Allow the mice to rest at the end of the full week of drug administration. Continue the weekly monitoring of the leukemia response by flow cytometry following steps 1.4 - 1.11.
      NOTE: At this point, the leukemia cells are expected to remain responsive to treatment. Consequently, when monitoring the leukemia, the percentage of hCD45+ in the mice should decrease and remain negative for a certain period.
2. Monitoring for R2 appearance
   1. Wait for relapse R2 to appear (hCD45+ cells ≥ 0.2%). When it appears, count the time elapsed between the end of treatment and the appearance of R2. Call this period Δt1.
   2. Proceed to a second full week of induction-type treatment administration. Follow the same guidelines as the first administration of this schedule.
   3. Allow mice to rest at the end of treatment. Continue leukemia monitoring.
3. Monitoring for R3 appearance
   1. Wait for relapse R3 to appear. Count the time in the same way as in step 3.2.1. Call it Δt2.
   2. Compare Δt1 and Δt2. Repeat the above steps N times to obtain multiple leukemia relapses (R1, R2...RN) if the Δt period remains the same. Stop treatment cycles once the Δt period shortens.
      NOTE: A decrease in Δt to relapse is an indication of drug resistance gained by the leukemia cells. This result reinforces the ability of the mice model of relapse to generate chemotherapy-resistant ALL cells.
   3. Recover leukemia cells as described in step 4 below. Store them alive by cryopreservation or by any preservative compatible with intended subsequent analyses.
      NOTE: The recommended event for the recovery of leukemia cells is up to 25% of hCD45+ in the peripheral blood, as described by recent literature¹⁶.

4. Leukemic cells recovery through organ harvesting

1. Mice euthanasia with an overdose of chemical anesthetics
   1. Euthanize mice by overdose of chemical anesthetics, followed by cervical dislocation for euthanasia confirmation as described below.
   2. Prepare a mixture containing three anesthetics diluted in sterile 1x PBS for deep anesthesia. Use 150 µL of commercial Acepromazine maleate, 50 µL of commercial Xylazine, 80 µL of commercial Ketamine, and 220 µL of PBS for each animal to be euthanized.
   3. Administer 500 µL of the prepared mixture by intraperitoneal injection. Follow the same restraint and injection guidelines as described in step 2.4.
   4. Under the effect of an overdose of chemical anesthetics, mice will lie down on the bottom of the cage. After a few minutes, observe if the mice have respiratory arrest. If so, wait for the respiratory effort to cease. To verify that the euthanasia procedure was effective, check for the presence of an involuntary reflex by applying pressure to one of the animal's hind legs. To confirm euthanasia, proceed to cervical dislocation.
2. Cervical dislocation
   1. Hold the animal with the rear of the body up against the cage feed grate. Use the index and thumb fingers of the left hand to form the shape of tweezers. Place the tweezer-shaped fingers directly under the animal's head. Press firmly against the cervical portion of the spine.
   2. Grasp the animal's tail with the right hand. Pull it back with a firm and steady motion until the animal's head is separated from the rest of its body.
      NOTE: Cervical dislocation is one of the accepted methods to confirm euthanasia^17,18,19. Other methods, such as continued exposure to CO₂ for at least 15 min after respiratory arrest, can be used as an alternative.
3. Animal preparation for organ harvesting
   1. Sanitize the mice by applying 70% alcohol to their entire body. Place the animal's body over a surgical platform for rodents with the ventral portion up. Shave the abdominal portion of the body.
      NOTE: A complete description of the surgical operating platform for rodents can be found in the Table of Materials.
4. Spleen harvesting
   1. Make a small incision with sterile surgical scissors and tweezers on the abdominal skin. Remove it from the abdominal area, exposing the peritoneum.
   2. Make a small incision in the peritoneum. Cut it until complete access to the organs located in the abdomen is gained.
   3. Locate the spleen, right below the animal's stomach. Remove the spleen. Place it in a recipient containing sterile 1x PBS for further processing.
5. Femur harvesting
   1. Proceed with opening the peritoneal cavity to gain access to the animal's hind legs.
   2. Locate the femur. Remove it by cutting at the joints that unite it with the pelvis bone and with the tibia and fibula. Do this process for both femur bones.
   3. Clean the bones by removing any remaining muscle, skin, or tissue. Place them in a recipient containing sterile 1x PBS for further processing. Discard the animal's carcass according to the guidelines of the Animal Ethics Committee.
      NOTE: Depending on the aims of the study, ALL cells could also be collected from the liver, central nervous system, thymus, lymph nodes, etc. In this manuscript, as an example, we describe the collection of ALL cells from the spleen and bone marrow.
6. Preparation for organ processing
   1. Gather one 50 mL sterile centrifuge tube, two 1 mL sterile syringes, one sterile 25 mm x 0.6 mm hypodermic needle, and two sterile 70 µm cell strainers.
7. Spleen processing
   1. Place one cell strainer on the top of an open 50 mL centrifuge tube. Put the spleen on the surface of the cell strainer. Macerate the organ with the flat part of the syringe plunger until no large portions are visible.
   2. Add sterile 1x PBS to the macerated spleen. Perform circular movements with the flat part of the syringe plunger to ensure cell filtration.
      NOTE: The PBS pouring can be done gradually; however, the final volume used cannot exceed 6 mL. Upon completion of the spleen processing, the cell strainer should be clear and devoid of any residual tissue. In the event of cell strainer saturation, it is possible to employ an alternative cell strainer. The contents of the initial strainer can be transferred into the second strainer, and the processing can then be continued. To access the tumor burden within this organ, a portion of the spleen can be separated for histopathological assessment.
8. Bone marrow processing
   1. Incorporate a new cell strainer for bone marrow processing into the already used centrifuge tube. Cut the edges of the femur bone until the bone marrow openings are accessible. Insert the hypodermic needle into a new syringe. Add 1 mL of sterile 1x PBS to the syringe.
      NOTE: Depending on the study's aims, ALL cells obtained from the spleen and bone marrow should be collected separately. In this case, a separate centrifuge tube should be used for each tissue. A portion of the femur can be separated for histopathological assessment to assess the tumor burden within this organ.
   2. Execute bone marrow flush over the cell strainer. Insert the needle tip into one side of the bone marrow opening. Push the syringe plunger with a firm and constant movement. Repeat the procedure with the other femur bone.
   3. Decouple the needle. Utilize the flat part of the syringe to macerate the flushed bone marrow. Add sterile 1x PBS to the macerated bone marrow. Perform circular movements with the flat part of the syringe plunger to ensure cell filtration. Upon completion of the processing procedure, the 50 mL centrifuge tube will contain a total volume of 10 mL, comprising a mixture of splenic and bone marrow cells that have been diluted in PBS.
      NOTE: The PBS pouring process can be executed gradually; however, it is imperative to ensure that the final volume utilized does not exceed 2 mL.

5. Isolation of leukemic cells by gradient centrifugation

1. Preparation for leukemic cells isolation
   1. Gather one 15 mL centrifuge tube, sterile disposable Pasteur pipettes, the 50 mL centrifuge tube containing the organs that have been processed, and the density gradient medium for lymphocyte isolation.
      NOTE: The medium facilitates the isolation of mononuclear cells by creating a density gradient through centrifugation. Mononuclear cells (lymphocytes, monocytes, and ALL cells) form a layer at the interface between the medium and the plasma.
2. Leukemic cells isolation
   1. Add 5 mL of the density gradient medium for lymphocyte isolation to the 15 mL centrifuge tube using a disposable Pasteur pipette. Gradually add the 10 mL of the mixture of splenic and bone marrow cells over the medium layer. Hold the tube at a shallow angle, around 30° or less, and slowly add the cell mixture along the tube wall so that the density medium layer is not perturbed.
      NOTE: Exercise caution during this process to ensure that the cells do not mix with the density medium. Disturbance during this process can lead to the failure of density gradient formation during centrifugation.
   2. Centrifuge the 15 mL tube at 420 x g on a swing bucket rotor, for 30 min, without acceleration and without break, to avoid disturbing the formed phases.
      NOTE: Leukemic cells are going to form a layer between the density medium and aqueous phases.
   3. Remove and discard the top aqueous phase using a sterile disposable Pasteur pipette. Collect the layer of leukemic cells formed with a sterile disposable Pasteur pipette. Transfer it to a new 15 mL centrifuge tube.
      NOTE: Do not worry if some of the lower-density medium layer is pipetted together with the leukemia cells.
3. Leukemic cells washing
   1. Add sterile 1x PBS to the tube containing the collected cells until the limit of 15 mL is reached.
   2. Centrifuge the cells at 300 x gfor 5 min. Restore maximum acceleration and braking. Remove and discard the supernatant. Resuspend the cells in new sterile cold 1x PBS.5.3.3.Proceed to cell and cell viability check. Follow standard cell count procedure guidelines¹³. The washed cells can be stained with anti-mouse CD45 antibody (mCD45), anti-human CD45 antibody (hCD45), and anti-human CD19 antibody (hCD19), followed by washing steps and data acquisition.
      NOTE: In case of processing the spleen cells and the bone marrow cells in different centrifuge tubes, a portion of these samples can be used to access tumor burden within these organs through flow cytometry.
4. Leukemic cells cryopreservation
   1. Pellet down the cells by centrifugation at 300 x g for 5 min. Discard the supernatant. Keep the cells on ice.
   2. Prepare a freezing medium consisting of 90% sterile cold fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). Use 1 mL of this solution for each cryopreservation vial.
   3. On ice, resuspend cells in the prepared solution at a concentration of 2 x 10⁷ cells/mL. Aliquot the cells into the cryopreservation tubes. Cryopreserve cells following standard cell culture cryopreservation guidelines²⁰.
      NOTE: These cells can be used for further analyses. Instead of freezing viable cells, one may also aliquot and preserve cells in different preservatives for subsequent DNA, RNA, protein, and metabolites analyses.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL  syringe	BD	#309628	Organ harvesting
10 mL sterile water for injection	Halex Istar Indústria Farmaceutica LTDA	https://www.medstoremaringa.com.br/produtos/agua-destilada-10ml-pack-c-5un-isofarma/	Drug reconstitution
10X RBC Lysis Solution	BioLegend	#420302	Mice blood sample processing
5 channels cell analyzer	BD Biosciences	658226R1	Flow Cytometer
70 uM Cell Strainer	Falcom	#352350	Organ harvesting
Acepromazine maleate Inj	Vetnil LTDA	https://vetnil.com.br/produto/acepran-r-1	Mice anesthesia
APC anti-human CD45 - isotype mouse IgG1 - HI30 clone	BioLegend	#304037	Lymphocyte staining
Asparaginase 10000 U	Aginase/medac	#88660100	Mice treatment
Brilliant-Violet 605 anti-mouse CD45 - isotype mouse IgG2 - 30-F11 clone	BioLegend	#103139	Lymphocyte staining
Daunorubicin	Farmarin	#116880025	Mice treatment
Dexamethasone	Aché	#00000066	Mice treatment
Dimethyl sulfoxide	Sigma-Aldrich	#589569	Lymphocyte cryopreservation
Disposable needle - 0.60 x 25 mm (23G x 1’’)	BD	#300388	Organ harvesting
Esterile disposable lancet	Yancheng Huida Medical Instruments	https://www.cralplast.com.br/produto/lanceta-manual/	Mice blood collection
Ethylenediaminetetracetic Acid	Life technologies	#15576-O28	EDTA solution
Fetal Bovine Serum	Cultilab	#F083	Lymphocyte cryopreservation
Flow Cytometry Software Analysis - Version 10.10.0	BD Biosciences	https://www.flowjo.com/	Flow cytometry analysis
Graph and Plotting Software Analysis - Version 9.5.1	Dotmatics	https://www.graphpad.com/features	Graph plotting and analysis
Insulin syringe with attached needle 1mL FX 12.7 mm X 0.33 mm	Descarpack	e0342101	Mice transplantation and drug administration
Ketamine Inj	Sespo Indústria e Comércio LTDA	https://www.bassopancotte.com.br/produto/dopalen-pecuaria/	Mice anesthesia
Multi-Functional Surgical Platform for rodents	Kent Scientific Corporation	https://www.kentscientific.com/products/surgisuite/	Mice surgical procedures
PE anti-human CD19 - isotype mouse IgG1 - HIB19 clone	BioLegend	#302208	Lymphocyte staining
Potassium chloride	Sigma-Aldrich	P3911-1KG	PBS solution
Potassium phosphate dibasic	Sigma-Aldrich	P0662-1KG	PBS solution
Reagent for lymphocyte isolation	Cytiva	#17-1440-02, pack of 6 × 100 mL	Lymphocyte isolation from harvested organs
Sodium chloride	Sigma-Aldrich	S9888-1kg	PBS solution
Sodium phosphate dibasic	Sigma-Aldrich	S9763-1KG	PBS solution
Sterile Anatomical Dissection Tweezeers - 12 cm ABC 0170	Lab Líder	120	Surgical mice procedures
Sterile Scissors 12 cm - ABC 0321	Lab Líder	136	Surgical mice procedures
Vincristine	Libbs	#7896094208483	Mice treatment
Xylazine Inj	Sespo Indústria e Comércio LTDA	https://www.bassopancotte.com.br/produto/anasedan-10-ml-bovinos-e-equinos/	Mice anesthesia