Novel single-cell technologies in acute myeloid leukemia research

Abstract

Acute myeloid leukemia (AML) is a lethal malignancy because patients who initially respond to chemotherapy eventually relapse with treatment refractory disease. Relapse is caused by leukemia stem cells (LSCs) that reestablish the disease through self-renewal. Self-renewal is the ability of a stem cell to produce copies of itself and give rise to progeny cells. Therefore, therapeutic strategies eradicating LSCs are essential to prevent relapse and achieve long-term remission in AML. AML is a heterogeneous disease both at phenotypic and genotypic levels, and this heterogeneity extends to LSCs. Classical studies in AML have aimed at characterization of the bulk tumor population, thereby masking cellular heterogeneity. Single-cell approaches provide a novel opportunity to elucidate molecular mechanisms in heterogeneous diseases such as AML. In recent years, major advancements in single-cell measurement systems have revolutionized our understanding of the pathophysiology of AML and enabled the characterization of LSCs. Identifying the molecular mechanisms critical to AML LSCs will aid in the development of targeted therapeutic strategies to combat this deadly disease.

Fig 1.

Analysis of mass cytometry data reveals functional heterogeneity in AML. Mll/AF9-NRAS^G12V murine AML cells^, stained with a panel of cell surface markers and intracellular signaling intermediates were analyzed by mass cytometry. (A) SPADE plot revealing the immunophenotypic architecture of cells. Each circle on the plot represents a group of cells with similar expression levels of the measured cell surface markers. The size of the circle represents the number of cells in each cluster. The color of the circles is representative of the level of CD11b of each cluster. (B) viSNE plot of murine AML cells retaining single-cell granularity. Each point on the plot represents a cell. Cells that are similar in all the measured dimensions are aligned closely together in the plot. In this plot, the color represents levels of CD11b. Both SPADE and viSNE plots can be colored according to each of the measured epitopes. CD11b^Low cells are indicated by a circle on the viSNE plot. These cells harbor the leukemia stem cell (LSC)-enriched compartment. (C) The color of the viSNE or SPADE plot can be changed to reflect the levels of any of the proteins measured. In this experiment, a panel of signaling intermediates were measured including pErk, pAkt, p4EBP1, pSTAT5, and total levels of Myc and β-catenin. Using CD11b, we gated on the LSC-enriched compartment in panel B. In panel C, the levels of pSTAT5 and Myc are displayed within this CD11b^Low population. This data allows us to conclude that CD11b^Low cells (enriched for LSCs) are high in Myc and low in pSTAT5.

Fig 2.

A diagrammatic representation summarizing the application of single-cell approaches to address different biological questions in AML.