Dynamic Quantitative Analytic Response of Human Natural Killer Cells at Single-Cell Resolution in B-cell Non-Hodgkin Lymphoma

Natural Killer (NK) cells are phenotypically and functionally diverse lymphocytes that recognize and kill cancer cells. The susceptibility of target cancer cells to NK cell-mediated cytotoxicity depends on the strength and balance of regulatory (activating/inhibitory) ligands expressed on target cell surface. We performed gene expression arrays to determine patterns of NK cell ligands associated with B-cell non-Hodgkin lymphoma (b-NHL). Microarray analyses revealed differential upregulation of a multitude of NK-activating and costimulatory ligands across varied lymphoma cell lines and b-NHL cells including ULBP1, CD72, CD48 and SLAMF6. To correlate genetic signatures with functional anti-lymphoma activity, we developed a dynamic and quantitative cytotoxicity assay in an integrated microfluidic droplet array. This assay facilitated the examination of transient interactions between NK cells and target lymphoma cells and also the functional heterogeneity in NK cell effector outcomes at single-cell level. Individual NK cells and target lymphoma cells were co-encapsulated in picoliter-volume droplets and imaged over time to quantify heterotypic cell-cell interaction. We identified significant variability in NK-lymphoma cell contact duration, frequency and subsequent cytolysis. The death of lymphoma cells undergoing single contact with NK cells occurred faster than cells that made multiple short contacts. NK cells also killed target cells in droplets via contact-independent mechanisms that partially relied on calcium-dependent processes and perforin secretion, but not on cytokines (IFN-γ or TNF-α). We extended this technique to characterize cytolysis of primary cells from b-NHL patients. Diffuse large B-cell lymphoma cells from two patients showed similar contact durations with NK cells; primary Burkitt lymphoma cells made longer contacts and were lysed at later times. We further tested the cytotoxic efficacy of NK-92, a continuously growing NK cell line being investigated as an anti-tumor therapy, using our droplet-based bioassay. NK-92 cells were found to be more efficient in killing b-NHL cells compared with primary/fresh NK cells, requiring shorter contacts for faster killing activity. Taken together, our combined genetic and microfluidic analysis demonstrate significant heterogeneity in the dynamic interaction and associated cytotoxicity between NK cellular therapy and b-NHL cells. For genome-wide expression profiling of SUDHL4, SUHDL10, EL-2 (also called DLBCL.1), EL-5 (also called DLBCL.2), and Raji cells were grown under normal conditions and Human HT-12 BeadArray chips (Illumina, San Diego, CA) were used. RNA was isolated using RNeasy Minikit (Qiagen), following instructions recommended supplied by the manufacturer. Purity and the yield of isolated RNA was determined using Bioanalyzer. These experiments were performed in biological triplicates. All replicate samples were run individually. Total RNA of 500ng per sample was amplified using AmbionTotalPrep, and 1.5ug of the product was loaded onto the chips. Following hybridization at 55C, the chips were washed and then scanned using the Illumina iScan System. The data was checked with GenomeStudio (Illumina) for quality control. Data were corrected through normalization with the housekeeping genes, quantile normalized, collapsed to genes from probes, then imported into MultiExperiment Viewer, MeV for analysis. Statistically significant genes were determined by applying a one-way ANOVA with an adjusted Bonferroni correction and false discovery rate (FDR) < 0.05 that resulted in a list of significant genes. ------------------------------------ This dataset does not include experimental samples