Single-Cell Mass Cytometry Reveals Phenotypic and Functional Heterogeneity In Acute Myeloid Leukemia At Diagnosis and In Remission
Authors: Han Lina , Zeng Zhihong , Qiu Peng , Jorgensen Jeffrey L. , Mak Duncan H , Burks Jared K , McQueen Teresa , Ravandi Farhad , Roboz Gail J , Kantarjian Hagop M. , Kornblau Steven M. , Guzman Monica L. , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is organized in a hierarchy with a rare population known as leukemia stem cells (LSC) capable of self-renewal and propagation of the disease. Characterization of the unique phenotypes and complex signaling pathways in LSCs that survive induction chemotherapy is essential for understanding of the mechanisms of chemoresistance and designing the strategies to eliminate residual leukemia clones.
In this study, we compared signaling profiles of distinct phenotypic AML subsets in paired bone marrow (BM) samples collected at diagnosis and after achieving the complete remission (CR). Cell surface characteristics and signaling pathways activated within sub-populations of AML samples were defined using the novel technology of time-of-flight mass cytometry (CyTOF) that has the ability to perform up to 100 mutiparameter assays in single cells (Bendall et al, Science 2011). First, we validated CyTOF measurements by performing cross-comparisons of surface markers and intracellular proteins measured in AML cells with traditional multi-parametric flow cytometry (FCM). Frequencies of CD123+CD99+ population within CD34+CD38- cells were 73.7%±1.8% and 78.5%±3.7% by CyTOF and FCM. Patterns of specific activation of the intracellular proteins pSTAT5, pERK1/2 and pAKT by GM-CSF, PMA and SCF, and inhibition by selective kinase inhibitors showed excellent cross-platform consistency between CyTOF, FCM and immunoblotting. Next, mononuclear cells of 5 paired AML (at diagnosis and in CR) and of 3 normal BM (NBM) were stained with 11 cell surface markers (CD34, CD38, CD123, CD99, CD45, CD33, CD117, CD7, CD4, CD90 and CD133) and 8 intracellular markers (p-4EBP1, p-NF¦ÊB, p-STAT3, p-AKT, p-mTOR, p-ERK, p-S6 and p-STAT5). A SPADE (spanning-tree progression analysis of density-normalized events) (Qiu et al, Nat Biotechnol. 2011) tree plot was generated, representing clustered expression of the cell-surface antigens. Boundaries and annotations of the AML cells were manually defined to represent distinct cell subsets (Figure 1). We used the pooled data from NBM samples, which showed identical patterns, as a reference. SPADE analysis revealed several subsets unique to the diagnostic AML samples, which were eliminated by chemotherapy; and phenotypically distinct subsets in diagnostic samples that persisted in CR. Notably, a subset defined by the “traditional LSC” markers (CD45dimCD34+CD38lowCD90-CD33-CD117+; annotation #2) was readily identified in diagnostic samples and was significantly reduced by induction chemotherapy in 2 of the 5 AML samples. In one of these samples we identified a distinct subset co-expressing LSC markers CD45dimCD34+CD38lowCD33-CD117-CD99lowCD133low (annotation #3) that was present in both diagnostic (1.1%) and CR (1.7%) BM; this subset may have contributed to the MRD detected by standard leukemia-associated immunophenotypes.Figure 1The tree plot was generated using 11 cell surface proteins in AML and NBM, and colored by the median intensity of individual markers (CD34 is shown). Phenotypes of each annotation are indicated.Figure 1. The tree plot was generated using 11 cell surface proteins in AML and NBM, and colored by the median intensity of individual markers (CD34 is shown). Phenotypes of each annotation are indicated.
We next investigated intracellular signaling pathways in antigen-defined AML subpopulations using CyTOF. Activation of p-AKT and pS6 showed similar pattern in subsets defined by annotations 1, 9 and 10 at diagnosis (Figure 2A), and was largely reduced in the CR BM. In turn, activation of p-4EBP1 and p-mTOR were observed in multiple subsets (#1-5 and 9-11) in all diagnostic AML samples, especially in a subset 1 characterized by the “Progenitor” phenotype, and remained heightened in the CR samples (Figure 2B).Figure 2The heat map of the average expression of intracellular proteins in selected populations from individual samples. (A) Each column represents individual sample, and each row reflects expression of a certain protein for each annotation. (B) Signaling pathways in annotation #1 in individual samples.Figure 2. The heat map of the average expression of intracellular proteins in selected populations from individual samples. (A) Each column represents individual sample, and each row reflects expression of a certain protein for each annotation. (B) Signaling pathways in annotation #1 in individual samples.
In summary, using CyTOF and SPADE, we characterized phenotype-specific intracellular signaling pathways in AML samples at diagnosis and in CR. Persistent activation of p-mTOR and p-4EBP1 are identified in the subpopulations of AML progenitors in CR, and may present the potentially targetable pathways in AML. The study is ongoing with prospective CyTOF analysis of a larger set of paired AML samples at diagnosis, CR and relapse coupled with the molecular analysis of the distinct subpopulations.

Disclosures:
No relevant conflicts of interest to declare.