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Summary

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.

Abstract

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.

Introduction

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 alrea....

Protocol

All samples were obtained with the informed consent of the children'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

  1. 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.
  2. Prepare 100 mL of RPMI medium.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
RPMI 1640 Medium, GlutaMAX SupplementGibco, ThermoFisher Scientific61870-010
Fetal Bovine SerumBioseraFB-1001/100
Antibiotic-Antimycotic (100X)Gibco, ThermoFisher Scientific15240-062
Sodium bicarbonateSigma-AldrichS5761-500G
D-(+) GlucoseSigma-AldrichG7021-100G
Oligomycin ASigma-Aldrich75351-5MG
2-Deoxy-D-glucoseSigma-AldrichD8375-1G
FCCPSigma-AldrichC2920-10MG
DMSOSigma-AldrichD8418-100ML
RotenoneSigma-AldrichR8875-1G
Antimycin A from Streptomyces sp.Sigma-AldrichA8674-25MG
Seahorse XF Base Medium, 100 mLAgilent Technologies103193-100
L-glutamine solution, 200 mMSigma-AldrichG7513-100ML
HEPES solution, 1 M, pH 7.0-7.6Sigma-AldrichH0887-100ML
Sodium pyruvateSigma-AldrichP5280-25G
Bovine Serum AlbuminSigma-AldrichA2153-10G
Ficoll-Paque PlusSigma-AldrichGE17-1440-02Density gradient medium
Seahorse XFp FluxPakAgilent Technologies103022-100
Corning™ Cell-Tak Cell and Tissue AdhesiveThermoFisher ScientificCB40240
Seahorse Analyzer XFpAgilent TechnologiesS7802A
Seahorse XFp Cell Culture MiniplateAgilent Technologies103025-100

References

  1. DeBerardinis, R. J., Lum, J. J., Hatzivassiliou, G., Thompson, C. B. The Biology of Cancer: Metabolic Reprogramming Fuels Cell Growth and Proliferation. Cell Metabolism. 7, 11-20 (2008).
  2. Ward, P. S., Thompson, C. B.

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