Name: assessment of the metabolic profile of primary leukemia cells
Uploaded: 2018-11-21T13:00:00
Description: Watch this Scientific Journal Video about Assessment of the Metabolic Profile of Primary Leukemia Cells at JoVE.com

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...

<ol>
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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 Streptomyces 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&#8482; 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...

Watch this Scientific Journal Video about Assessment of the Metabolic Profile of Primary Leukemia Cells at JoVE.com

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 ...

This method can help answer key questions regarding metabolic pathway setup in leukemia cells. The main advantage of this technique is that it allows measurement of the metabolism of primary leukemia cells in real time in live cells. Begin by diluting a bone marrow sample obtained from a leukemia patient in PBS at a one to one ratio.Carefully, layer six milliliters of the diluted bone marrow sample over six milliliters of freshly prepared density gradient medium in a 15 milliliter conical tube and separate the cells by density gradient centrifugation. Use a Pasteur pipet to carefully transfer the interface layer of mononuclear cells to a new 50 milliliter conical tube containing five milliliters of PBS for a centrifugation wash. Then, resuspend the mononuclear cell pellet in two milliliters of sterile PBS for counting.Dilute the cells to a three times 10 to the seven cells per milliliter concentration. And add one milliliter of cells to each of two T75 flasks containing 20 milliliters of RPMI medium for a 16 to 24 hour incubation at 37 degrees Celsius and 5%CO2. Meanwhile to prepare the plates for extracellular flux analysis, add 12.5 microliters of cell adhesive solution into each well of two, eight well extracellular flux analyzer plates.After 20 minutes, aspirate the cell adhesive and wash each well two times with 200 microliters of sterile water per wash. After the second wash, leave the plates in the hood until the wells are dry. And place the sensor cartridge upside down on the lab bench.Separate the utility plate and the sensor cartridge of the flux analyzer. And fill each well of the utility plate with 200 microliters of calibrant and each moat around the outside of the wells with 400 microliters of calibrant. Return the sensor cartridge to the utility plate that now contains the calibrant and place the cartridge assembly in a humidified 37 degrees Celsius incubator without CO2 overnight.Then turn on the extracellular flux analyzer and let it warm to 37 degrees Celsius overnight. The next morning, transfer the cells from the flask to 50 milliliter conical tubes for centrifugation. And resuspend the pellets in one milliliter of the appropriate experimental medium for counting.Resuspend the cells at four times 10 to the six cells in 400 microliters of experimental medium concentration. And add 50 microliters of cells into wells B through G of the flux analyzer plate. And 180 microliters of the experimental medium into wells A and H as the background control wells.After centrifugation slowly and carefully add 130 microliters of the experimental medium to wells B through G.And visually confirm stable adherence of the cells to the bottom of each well under the microscope. Then return the flux analysis plate to the humidified 37 degrees Celsius incubator without CO2 for 30 minutes. 20 minutes before the end of the incubation, load the compounds into the appropriate injector ports of the cartridge according to the experimental protocol as indicated in the table.Then set up the appropriate extracellular flux analysis program. Start the program and replace the calibrant plate with the assay plate when prompted. In a glycolysis stress test, only basal medium is used so that the cells are deprived of nutrients.The first parameter obtained is the basal acidification which should reflect the amount of glucose stored in cells. After the first injection, the extracellular acidification rate is increased as the cells utilize the glucose and can ferment the glucose to lactate. The oligomycin A in the second injection inhibits ATP synthase and thus directs the cells to produce ATP mainly by a glycolysis causing further elevation of the extracellular acidification rate.While the injection of 2-Deoxy-D-glucose completely inhibits glycolysis and the extracellular acidification rate drops. In the cell mito stress test, a medium supplemented with glutamine and glucose is used so that the cells are not deprived of all nutrients. The basal respiration parameter reflects their basal metabolic state.After the first injection with oligomycin A, the cells inhibit mitochondrial respiration and switch to glycolysis which is represented as a decrease in the oxygen consumption rate. The second and third injections of FCCP however, uncouple the ATP production from the respiration so that the cells now consume oxygen at a maximal rate. And the oxygen consumption rate rises to its highest value.The last injection of the rotenone and antimycin A mixture completely inhibits the mitochondrial respiration decreasing the oxygen consumption rate to almost zero. While attempting this procedure, it's important to assess the percentage of blasts in the sample to ensure that the metabolic parameters of the leukemiablasts only are measured. When implementing this method, a careful optimization has to be applied to cultivation conditions and data normalization.

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