We would like to thank the Czech Pediatric Hematology Centers. This work was supported by the Grant of Ministry of Health (NV15-28848A), by Ministry of Health of Czech Republic, University Hospital Motol, Prague, Czech Republic 00064203 and by Ministry of Education, Youth and Sports NPU I nr.LO1604.

All samples were obtained with the informed consent of the children&#39;s parents or guardians and approval of Ethical committee of Charles University in Prague, Czech Republic, the study no. NV15-28848A.
1. Preparation of Reagents
<ol>
	<li>Prepare 500 mL of PBS by dissolving 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.47 mM KH2PO4, in ddH2O. Adjust the pH to 7.4 with HCl. Sterilize by autoclaving.</li>
	<li>Prepare 100 mL of RPMI medium: RPMI-1640 medium with L-Alanyl-Glutamine supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 &#956;g/mL).</li>
	<li>Prepare 50 mL of 0.1 M NaHCO3 in distilled water. Adjust the pH to 8.1, filter sterilize (0.22 &#956;m) and store at 4 &#176;C. 
	NOTE: For two 8-well extracellular flux analyzer plates, prepare 250 &#956;L of 0.1 M NaHCO3 pH 8.1.</li>
	<li>Prepare 1 mL of 2 M D-glucose in distilled water. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mM Oligomycin A in ethanol. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 250 &#956;L of 1 M 2-deoxy-D-glucose (2-DG) in Minimal DMEM (Dulbecco&#39;s Modified Eagle&#39;s medium). Warm to 37 &#176;C and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C). Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mM FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in DMSO. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mM Rotenone in ethanol. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Prepare 100 &#956;L of 1 mg/mL Antimycin A in ethanol. Filter sterilize (0.22 &#956;m) and store at -20 &#176;C.</li>
	<li>Just before the use, prepare 10 mL of Glycolysis stress test medium. Warm Minimal DMEM to 37 &#176;C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C).</li>
	<li>Prior to the use, prepare 10 mL of Cell Mito stress test medium with BSA. Supplement Minimal DMEM with 2 mM L-glutamine, 10 mM D-glucose, 1 mM HEPES (pH 7.4), 1 mM pyruvate and 0.1 % BSA (bovine serum albumin). Warm to 37 &#176;C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C).</li>
	<li>Prior to the use, prepare 10 mL of Cell Mito stress test medium without BSA. Supplement Minimal DMEM with 2 mM L-glutamine, 10 mM D-glucose,1 mM HEPES (pH 7.4) and 1 mM pyruvate. Warm Cell Mito stress test medium without BSA to 37 &#176;C in a water bath and adjust the pH to 7.4 (carry out pH measurement at 37 &#176;C). 
	NOTE: BSA is added to the Cell Mito stress test medium because the cells respond better to FCCP when the medium is supplemented with BSA (different conditions were tested). Cell Mito stress test medium without BSA is used for loading the ports as the manufacturer does not recommend using BSA in the medium.</li>
</ol>
2. Isolation of Mononuclear Cells from Bone Marrow
NOTE: Ideally, measurement of metabolic state of primary leukemia cells should start immediately after bone marrow collection and cell isolation. Nevertheless, relevant data could be also obtained from cells isolated after transportation from other hematology centers in the Czech Republic. Perform all sub-steps in a sterile tissue culture hood.
<ol>
	<li>Warm up PBS and the density gradient medium to room temperature.</li>
	<li>Dilute the bone marrow sample of leukemia patient with PBS, in a ratio of 1:1. Make sure that the sample contains at least 80% of leukemic blasts.</li>
	<li>Determine the percentage of blasts by flow cytometry using specific CD markers for immunophenotype characterization of cell types. After density gradient separation, detect nucleic acid positive events with a nuclear dye in order to establish the overall cell count.</li>
	<li>Then, determine leukemia cells using specific CD markers for each type of leukemia: B-ALL (CD19, CD45), T-ALL (CD3, CD4, CD8, CD5, CD7, CD99) and AML (CD45, CD33, and specific myeloid markers)24. Divide the number of leukemia cells positive for specific CD markers by all nuclear cells to determine the percentage of leukemia blast cells. 
	NOTE: The bone marrow has to be collected into the tubes with anticoagulants.</li>
	<li>Carefully layer 6 mL of diluted bone marrow sample over 6 mL of the density gradient medium in a 15 mL conical tube. Centrifuge at 400 x g for 35 min at 4 &#176;C in a swinging-bucket rotor without the brake. 
	NOTE: Larger volume of the sample could be divided into more aliquots or 50 mL conical tube can be used with the larger amount of density gradient medium.</li>
	<li>Using a Pasteur pipette, carefully transfer the interphase layer which consists of mononuclear cells (Figure 1) to a new 50 mL conical tube with 5 mL of PBS. Centrifuge at 400 &#215; g for 10 min at 4 &#176;C in a swinging-bucket rotor. 
	NOTE: Transfer all mononuclear cell layers into a single 50 mL conical tube with 5 mL of PBS.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 2 mL of sterile PBS. Count the cells using a hemocytometer. 
	NOTE: The number of leukemia cells in one mL of aspirated bone marrow differs significantly among patients25.</li>
</ol>
3. Overnight Cultivation of Mononuclear Cells
NOTE: Perform all sub-steps in a sterile tissue culture hood.
<ol>
	<li>Prepare two T75 flasks with 20 mL of RPMI. To each flask, add 30 x 106 isolated mononuclear cells. Incubate the cells with the flask standing up for 16&#8211;24 h at 5% CO2 and 37 &#176;C.</li>
</ol>
4. Preparation of Cell Adhesive-coated Plates
<ol>
	<li>Coat two 8-well extracellular flux analyzer plates. 
	NOTE: Perform the coating in a sterile tissue culture hood.</li>
	<li>Add 2.2 &#956;L of cell adhesive(density: 2.54 mg/ml) to 250 &#956;L of 0.1 M NaHCO3, pH 8.1, and pipette immediately 12.5 &#956;L of the solution into each well. 
	NOTE: Cell adhesive stock solutions can differ in their density, adjust the volume added to NaHCO3 accordingly.</li>
	<li>Let the plates sit in the hood for about 20 min, then aspirate the cell adhesive and wash each well twice using 200 &#956;L of sterile water. Let it sit in the hood with the lid open until wells are dry.</li>
	<li>Use the plates right away or save up to 1 week at 4 &#176;C with the rim wrapped in paraffin film to avoid condensation. Ensure that the plates are warmed up to room temperature (for about 20 min) in the hood before seeding the cells.</li>
</ol>
5. Hydration of Sensor Cartridge
NOTE: Hydrate two 8-well extracellular flux analyzer cartridges.
<ol>
	<li>Separate the utility plate and the sensor cartridge. Place the sensor cartridge upside down on the lab bench.</li>
	<li>Fill each well of the utility plate with 200 &#956;L calibrant. Fill each moat around the outside of the wells with 400 &#956;L of calibrant.</li>
	<li>Return the sensor cartridge to the utility plate that now contains the calibrant.</li>
	<li>Place the cartridge assembly in a humidified, non-CO2, 37 &#176;C incubator overnight.</li>
	<li>Turn on the extracellular flux analyzer and let it warm to 37 &#176;C overnight.</li>
</ol>
6. Seeding Cells in Cell Adhesive-coated Plates
NOTE: For Glycolysis stress test, use Glycolysis stress test medium. For Cell Mito stress test, use Cell Mito stress test medium with BSA.
<ol>
	<li>Seed cells for the Glycolysis stress test and the Cell Mito stress test in separate plates. For each test, use cells from one flask with overnight culture.</li>
	<li>Centrifuge cells at 200 x g for 5 min at room temperature. Resuspend cells in 1 mL of the appropriate medium and count them.</li>
	<li>Add 4 x 106 of live cells to the final volume of 400 &#956;L (use appropriate medium).</li>
	<li>Plate 50 &#956;L of the cell suspension into wells B-G. Ensure 500,000 cells are seeded in one well. 
	NOTE: It is crucial to seed exactly 500,000 cells per well as no other normalization is performed. That way, the results from different patients can be compared. The optimal number of replicates is six, as is described here. Using less replicates is not recommended since primary cells could sometimes behave erroneously.</li>
	<li>Add 180 &#956;L of the appropriate medium into wells A and H (these wells will serve as a background correction).</li>
	<li>Centrifuge the plate at 400 x g for 5 min at room temperature with brake set to 1.</li>
	<li>Add 130 &#956;L of appropriate medium to wells B-G in two 8-well extracellular flux analyzer plates slowly and carefully. Visually confirm that the cells are stably adhered to the bottom of the wells by viewing under the microscope.</li>
	<li>Place the plate into a humidified, non-CO2, 37 &#176;C incubator for 30 min.</li>
</ol>
7. Loading the Sensor Cartridge
<ol>
	<li>For the Glycolysis stress test, prepare 250 &#956;L each of 100 mM glucose, 20 &#956;M Oligomycin A and 1 M 2-DG, all in Glycolysis stress test medium.</li>
	<li>For the Cell Mito stress test, prepare 250 &#956;L each of 20 &#956;M Oligomycin A, 15 &#956;M FCCP, 30 &#956;M FCCP and a mixture of 10 &#956;M Rotenone and 10 &#956;g/ml Antimycin A, all in Cell Mito stress test medium without BSA. 
	NOTE: Injecting two concentrations of FCCP in one assay is recommended since there is not enough patients&#39; material for the FCCP titration. Nevertheless, the concentrations should be determined by the researcher.</li>
	<li>Load the compounds into the appropriate injector ports of the cartridge as follows (Table 1):</li>
</ol>
8. Setting Up the Program
<ol>
	<li>For the Glycolysis stress test, set up the program as described in Table 2.</li>
	<li>For the Cell Mito stress test, set up the program as described in Table 3.</li>
	<li>Start the program. Replace the calibrant plate with the assay plate (when prompted).</li>
</ol>
9. Evaluation and Interpretation of the Results
<ol>
	<li>In the Glycolysis stress test results, subtract the lowest ECAR value after 2-DG injection from all other ECAR values. 
	NOTE: This lowest value represents the non-glycolytic acidification. Usually, the lowest value is from the 4th measurement.</li>
	<li>Calculate Basal acidification, Glycolysis, Maximal glycolysis and Glycolytic reserve parameters from glycolytic function (Figure 2A). After subtracting the lowest ECAR value, calculate Basal acidification as a mean of ECAR from the first three measurement points (omit the first ECAR value if it significantly differs from the other two), calculate Glycolysis as a mean of ECAR from three measurement points after glucose injection and calculate Maximal glycolysis as a mean of ECAR from three measurement points after Oligomycin A injection. Calculate Glycolytic reserve as Maximal glycolysis minus Glycolysis. 
	NOTE: Alternatively, for Maximal glycolysis calculation, use the highest ECAR value from the three points measured.</li>
	<li>In the Cell Mito stress test results, subtract the lowest OCR value after Rotenone/Antimycin A injection from all other OCR values.</li>
	<li>Calculate Basal respiration, ATP production, Maximal respiration and Spare capacity parameters from mitochondrial function (Figure 2B). After subtracting the lowest OCR value, calculate Basal respiration as a mean of OCR from the first three measurement points. 
	NOTE: Maximal respiration is the highest OCR value after FCCP injection.</li>
	<li>For ATP production calculation, subtract the mean of the three OCR measurement points after Oligomycin A injection from Basal respiration. Calculate Spare capacity as the Maximal respiration minus the Basal respiration. 
	NOTE: Always calculate the Maximal respiration from the highest OCR value, regardless of the FCCP concentration used.</li>
</ol>

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The authors have nothing to disclose.

The above-described protocol allows for the measurement of the metabolic activity assessed by OCR and ECAR values in primary leukemic blasts derived from patients with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). The advantage of measurement using an extracellular flux analyzer is that it enables the detection of metabolic profile in the real time in the live cells. Essentially, every step in the provided protocol could be adjusted depending on the cell type one plans to study. Here, we will discus...

The metabolic profile is one of the main characteristics of cells and altered bioenergetics are now considered one of the hallmarks of cancer1,2,3. Moreover, changes in the metabolic setup could be used in the treatment of cancer by targeting signal transduction pathways or enzymatic machinery of cancer cells4,5,6. Knowing the metabolic predisposition of cancer cells is thus an advantage and can help improve the current therapy.
There are a plenty of already established methods which can assess the metabolic activity of cells in culture. Regarding glycolysis, glucose uptake can be measured by the radioactive labeling, using 2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) or extracellular lactate levels measured enzymatically7,8. Fatty acid oxidation rate is another metabolic parameter measured by isotopically labeled palmitate9,10. Oxygen consumption rate is a method widely-used for determining mitochondrial activity in cells11,12, together with the mitochondrial membrane potential evaluation13,14, ATP/ADP (adenosine 5&#8242;-triphosphate/Adenosine 5&#8242;-diphosphate) ratio measurement15 or total intracellular ATP measurement16. Signaling pathways known to regulate metabolic processes could be determined by protein quantifications and can improve the understanding of metabolic measurements17,18,19.
However, all these methods measure only one or, in the best scenario, a few metabolic parameters in one sample simultaneously. Importantly, simultaneous measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) can be achieved by the extracellular flux analysis by, for example, Seahorse XFp Analyzer. OCR is an indicator of mitochondrial respiration and ECAR is mainly the result of glycolysis (we cannot ignore CO2 production possibly elevating ECAR of cells with high oxidative phosphorylation activity)20. So far, various cell types have been studied using these analyzers21,22,23.
Here we describe the protocol for the extracellular flux analysis of primary blasts (leukemia cells derived from the immature hematopoietic stage) from leukemia patients. To the best of our knowledge, a specific protocol for primary blasts is not available yet.

<table><tbody><tr><td>RPMI 1640 Medium, GlutaMAX Supplement</td><td>Gibco, ThermoFisher Scientific</td><td>61870-010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Biosera</td><td>FB-1001/100</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Gibco, ThermoFisher Scientific</td><td>15240-062</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761-500G</td><td></td></tr><tr><td>D-(+) Glucose</td><td>Sigma-Aldrich</td><td>G7021-100G</td><td></td></tr><tr><td>Oligomycin A</td><td>Sigma-Aldrich</td><td>75351-5MG</td><td></td></tr><tr><td>2-Deoxy-D-glucose</td><td>Sigma-Aldrich</td><td>D8375-1G</td><td></td></tr><tr><td>FCCP</td><td>Sigma-Aldrich</td><td>C2920-10MG</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418-100ML</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875-1G</td><td></td></tr><tr><td>Antimycin A from &lt;em&gt;Streptomyces&lt;/em&gt; sp.</td><td>Sigma-Aldrich</td><td>A8674-25MG</td><td></td></tr><tr><td>Seahorse XF Base Medium, 100 mL</td><td>Agilent Technologies</td><td>103193-100</td><td></td></tr><tr><td>L-glutamine solution, 200 mM</td><td>Sigma-Aldrich</td><td>G7513-100ML</td><td></td></tr><tr><td>HEPES solution, 1 M, pH 7.0-7.6</td><td>Sigma-Aldrich</td><td>H0887-100ML</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P5280-25G</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A2153-10G</td><td></td></tr><tr><td>Ficoll-Paque Plus</td><td>Sigma-Aldrich</td><td>GE17-1440-02</td><td>Density gradient medium</td></tr><tr><td>Seahorse XFp FluxPak</td><td>Agilent Technologies</td><td>103022-100</td><td></td></tr><tr><td>Corning&amp;trade; Cell-Tak Cell and Tissue Adhesive</td><td>ThermoFisher Scientific</td><td>CB40240</td><td></td></tr><tr><td>Seahorse Analyzer XFp</td><td>Agilent Technologies</td><td>S7802A</td><td></td></tr><tr><td>Seahorse XFp Cell Culture Miniplate</td><td>Agilent Technologies</td><td>103025-100</td><td></td></tr></tbody></table>

assessment of the metabolic profile of primary leukemia cells

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic pathways is tested in many types of tumors. Thus, characterization of cancer cell metabolic setup is inevitable in order to target the correct pathway to improve the overall outcome of patients. Unfortunately, in a majority of cancers, the malignant cells are quite difficult to obtain in higher numbers and the tissue biopsy is required. Leukemia is an exception, where a sufficient number of leukemic cells can be isolated from the bone marrow. Here, we provide a detailed protocol for the isolation of leukemic cells from leukemia patients bone marrow and subsequent analysis of their metabolic state using extracellular flux analyzer. Leukemic cells are isolated by the density gradient, which does not affect their viability. The next cultivation step helps them to regenerate, thus the metabolic state measured is the state of cells in optimal conditions. This protocol allows achieving consistent, well-standardized results, which could be used for the personalized therapy.

Figure 3 shows the curves after Glycolysis stress test and Cell Mito stress test measurements of leukemic blasts from the BCP-ALL (B-cell precursor acute lymphoblastic leukemia) and AML (acute myeloid leukemia) patients. The calculation of metabolic parameters from these measurements is also indicated. 500,000 cells per well were seeded and all measurements were done in hexaplicates.
In the Glycolys...

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Assessment of the Metabolic Profile of Primary Leukemia Cells

Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

The metabolic requirement of cancer cells can negatively influence survival and treatment efficacy. Nowadays, pharmaceutical targeting of metabolic ...

assessment-of-the-metabolic-profile-of-primary-leukemia-cells

Research

JoVE Journal

Cancer Research

10.3K Views.  Charles University, Prague. Here we present a protocol for the isolation of leukemic cells from leukemia patients bone marrow and analysis of their metabolic state. Assessment of the metabolic profile of primary leukemia cells could help to better characterize the demand of primary cells and could lead up to more personalized medicine.

Video: Assessment of the Metabolic Profile of Primary Leukemia Cells

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Chemistry

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

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

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

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.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Medicine

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

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

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

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.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Immunology and Infection

Modeling Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Low-Input Metabolic Studies in Mice Cell Types

Targeted Metabolomics on Rare Primary Cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Author Spotlight: Advancing Metabolomics Analysis of Rare Hematopoietic Stem Cells 

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

Assessment of the Metabolic Profile of Primary Leukemia Cells

Summary

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Chapters in this video

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

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

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

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

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Modeling Chemotherapy Resistant Leukemia In Vitro

Targeted Metabolomics on Rare Primary Cells