Abstract SY14-01: 34+ parameter single-cell mass cytometry of differential immune and drug responses across healthy human hematopoiesis and related malignancies

Abstract

Abstract Fluorescence flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. Using this, intracellular assays of signaling systems can be correlated to functional subsets of cells in complex populations based on active kinase states or other signaling junctions. Such correlations could be important to distinguish changes in signaling status that arise in rare cell subsets during environmental activation or pathogenesis. Historically, fluorescence spectral overlap has stymied the use of an increasing number of phenotypic parameters required to delineate an array of rare cell populations, thus limiting the application of this single-cell approach in studying complex diseases. Using a next-generation single-cell ‘mass cytometry’ platform we avoid these caveats, quantifying 34 parameters simultaneously with no spectral overlap (e.g., 31 antibodies, viability, DNA content and relative cell size). In healthy human blood and bone marrow, we identified cell subsets spanning the hematopoietic hierarchy using a panel of up to 31 surface markers. Additionally, 18 simultaneous functional markers and a battery of ex vivo stimuli and inhibitors were used to interrogate signaling. A novel algorithm for unsupervised high-dimensional clustering of single-cell data revealed a continuum of phenotypically contiguous subpopulations. This continuum was demarcated by surface marker expression, but paralleled by gradual changes in intracellular signaling architecture. These results have exposed unappreciated layers of human hematopoietic organization, and provide an opportunity to reevaluate diseases and pharmacological therapeutics as specific perturbations to this inherent order. Working from a 34-parameter template of normal hematopoiesis, we now are now focused on further dissecting the biology and improving risk stratification in hematopoietic malignancies. The application of these multidimensional approaches to human acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) samples will be presented. The simultaneous measurement of surface marker, cell cycle, apoptosis, and intracellular signaling parameters has revealed substantial heterogeneity in the blast populations of these diseases. This information is used to provide a high-content view of in vitro drug activity in blast subpopulations. Leveraging the wide variability between human individuals, we have integrated single-cell data from multiple individuals to construct a system-level correlation map of seemingly independent immune compartments. Specifically, we reframe the healthy immune system as a homeostatic network of immune parameters including cell frequency, surface marker intensity, and intracellular signaling capacity. Chronic diseases such as rheumatoid arthritis and prolonged bacterial infection fundamentally disrupt this balance, causing measurable and pathologically relevant shifts in the normal homeostatic balance. Taken together, these early successes point toward a future of actionable clinical information based on single-cell assays for diverse hematological malignancies and autoimmune disorders. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY14-01. doi:10.1158/1538-7445.AM2011-SY14-01