Miniaturized High-Throughput Multiparameter Flow Cytometry Assays Measuring In Vitro Human Dendritic Cell Maturation and T-Cell Activation in Mixed Lymphocyte Reactions.

Enhancing antitumor activities of the human immune system is a clinically proven approach with the advent of monoclonal antibodies recognizing programmed cell death protein-1 (PD1) receptors on immune cell surfaces. Historically, using flow cytometry as a means to assess next-generation agent activities was underused, largely due to limits on cell number and assay sensitivity. Here, we leveraged an IntelliCyt high-throughput flow cytometry platform to monitor human dendritic cell maturation and lymphocyte proliferation in mixed lymphocyte reactions. Specifically, we established flow cytometry-based immunophenotyping and screening methodologies capable of measuring T-cell activation as a result of cell-associated antigens presented on dendritic cell surfaces, as indicated by cell proliferation, cytokine secretion, and surface marker expression. Together, the overall novelty of this 384-well platform is its capability to measure multiple functional readouts in one well and consistently evaluate large numbers of compounds in a single study, as well as its ability to show increased assay sensitivity requiring considerably fewer primary cells and less reagents compared to more traditional 96-well flow cytometry methods.

Establishing a high-throughput and automated cancer cell proliferation panel for oncology lead optimization.

Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification, and optimization, as well as determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency. Here we describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines were selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance were developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) was applied to analyze the data and generate data reports. Using tool compounds, we have shown that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, we have demonstrated that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, to drive cellular structure-activity relationships, and to profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization.