IS5-1 - Identification and Characterization of Acute Myeloid Leukemia-Stem Cells by Single-Cell Gene-Expression Profile

Abstract

ABSTRACT While the identification of cancer stem cells as therapeutic targets is now actively being pursued in a variety of human malignancies, leukemic stem cells (LSCs) in acute myeloid leukemia (AML) are a paradigm of such a strategy. However, significant improvements in outcome based on these insights have not been forthcoming, because the molecular basis to distinguish the properties of LSCs from those of normal hematopoietic stem cells (HSCs) remains largely unknown. Meanwhile, current advances in single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. To characterize biological subtypes of LSCs in AML, in this study, we systematically analyzed the cDNA profiles of a large number of single LSCs. We first carried out the comparative DNA microarray analysis with primary leukemic cells and reconstituted cells in xenograft, and extracted 22 intrinsic genes from 5,599 genes, of which expression was significantly up-regulated in reconstituted cells, as the markers of high grade, aggressive AML. Among them, Evi1 and MN1, both previously identified potential poor prognostic factors in AML, were included. Next, we isolated BM CD34 + 38- LSCs from refractory AML patients, randomly picked up single cells, and amplified a total of 96 single-cell cDNA samples from each patient, as well as CD34 + 38- cells derived from a healthy volunteer to obtain information from normal HSCs. By analyzing expression-patterning of the 22 identified genes in each cell, we have identified a set of genes, of which expression pattern was significantly correlated with that of Evi1. This correlation was not observed in their normal counterparts, indicating that it was specific for AML LSCs. Furthermore, in one case, who developed AML from myelodysplastic syndrome (MDS), the hierarchical cluster based on the molecular signatures was identified only in LSCs at AML stage, but not at MDS. These results suggest that single-cell gene-expression profiling would allows us to classify AML-LSCs and provide new insights into functional properties of LSCs. Currently, we are comprehensively evaluating the expression of surface molecules on AML LSCs, and would like to disclose the clinical practicality of this methodology in our presentation.