Thin Layer Chromatography Goes Ultrasmall to AssaySphingosine Kinase Activation in Single Primary Leukemic Cells

Cell-to-cell heterogeneity in lipid signaling underlies variations in response and recurrence for many cancers, including leukemias. A highly parallel, miniaturized thin-layer chromatographic platform capable of assaying single cells was developed. Ultrasmall volumes (50 pL) of standard fluorescent lipids were separated with excellent repeatability, reproducibility, and limits of detection. Sphingosine-cyanine 5 (Sph-Cy5) was loaded into cells, and the single-cell contents were separated to identify Sph-Cy5 and two metabolites, Sph-1-phosphate-Cy5 (S1P-Cy5) and hexadecanoic acid Cy5 (HA-Cy5). In leukemic cells, the CD34+ blast cells demonstrated significantly greater conversion of Sph-Cy5 to its phosphorylated form compared to that of the CD34- cells. After treatment with a sphingosine kinase (SphK) inhibitor, the level of formation of S1P-Cy5 remained significantly greater for the inhibited CD34+ cells relative to that of the inhibited CD34- cells. Over 1200 single cells were rapidly assayed using 8 chips within 4 h. Sphingosine kinase activity in the CD34+ blast cells of 3 patients with acute myeloid leukemia was assayed with and without inhibitors. The patient cells displayed intertumor and intratumor heterogeneity, and subsets of cells with distinct enzymatic activities and products, highlighting the diversity of the cells within a clinical sample and between patients.