Malignant Cells in the Marrow and Blood of Patients with Hematopoietic Malignancies Have Higher Mitochondrial Membrane Potential as Measured with the Potentiometric Dye JC-1.

Abstract Background: Immunophenotyping is an important method to define hematopoietic malignancies, but used alone, its ability to provide functional information about the malignant cells is limited. JC-1 is a fluorescent dye used to measure mitochondrial membrane potential in cells. We have developed a flow cytometric assay using JC-1 in conjunction with standard cell surface markers to characterize clinical samples from patients with hematologic malignancies. Methods: Ficoll-purified cells derived from the blood or marrow were stained with JC-1, and analyzed at 530 nm and 590nm to assess mitochondrial mass and mitochondrial membrane potential, respectively. Malignant and normal cell populations were assigned and gated by analysis of forward scatter and side scatter. Gating in this manner identified energetically discrete cell populations confirmed by back gating analysis. Results: To date we have measured the mitochondrial membrane potential from the bone marrow or peripheral blood of 3 normal individuals and 22 patients with acute and chronic leukemias and lymphoma involving the marrow. Samples have been analyzed from AML (6), ALL (3), CML- chronic phase (2), CLL (5), mantle cell lymphoma (3), diffuse large B-cell lymphoma (1) and acute biphenotypic leukemia (2). Compared with samples derived from normal individuals, malignant cells have a different energetic signature, and in nearly all instances, a substantially higher mitochondrial membrane potential. The mitochondrial membrane potential of malignant cells was 5.5 times higher (range 2.15–12) than normal cells within the same samples in 19 of 20 specimens (given the overlap in cell populations, CML samples were excluded and 1 CLL sample had a mitochondrial membrane potential that was the same as the normal population). Mitochondrial membrane potential is also higher in malignant cells compared with peripheral blood and marrow cells from normal individuals. While cells derived from patients with acute leukemias appear to be energetically homogeneous, cells from both patients with CML and 4 of 5 patients with CLL had at least 2 energetically discrete subpopulations despite being cytogenetically homogeneous (in the case of the CML samples) or immunophenotypically homogenous (in the case of the CLL samples). Conclusions: JC-1 staining can be performed reliably in concert with immunocytochemistry and provides insight into the metabolism of malignant hematopoietic cells. Preliminary data indicate that malignant cells from patients with leukemia and lymphoma have a high mitochondrial membrane potential which may reflect altered oxidative metabolism. This may serve as a metabolic signature for monitoring minimal residual disease or assessing the effect of therapeutic agents on functional status of malignant populations.