Single Cell Methods for Bioeffector Discovery and Analysis

PROJECT SUMMARY Natural product therapeutics remain critically important in the treatment of cancer, with most patients diagnosed with cancer receiving natural product-based chemotherapy, such as microtubule disrupting agents and DNA intercalators. Select chemotherapeutic small molecules, the majority of which are natural products, have an additional therapeutic benefit in their ability to stimulate a productive form of natural immunity against the cancer cells that they injure or kill. For example, chemically induced damage associated molecular pattern expression can recruit antigen presenting cells to phagocytose damaged cancer cells and display cancer cell antigens to prime and activate T cells for adaptive immunity. Notably however, natural product induced anti-tumor immunity properties of these select compounds were only discovered subsequent to their clinical application. We hypothesize that the pathways by which such immunogenic natural products injure and kill cancer cells determines the production of damage associated molecular patterns and ensuing innate and adaptive immune responses against treated cells. We propose to develop and apply a high throughput, multiplexed, single cell chemical biology assay platform for the discovery of chemically induced antitumor immunity. Using this system, we will map the associations between regulated cell death and injury signaling in treated cells to functional cellular markers of immunogenicity for several classes of known and new natural products. This goal will be accomplished through three aims: (1) Discover and characterize secondary metabolites via regulated cell death and injury cytometric phenotypes; (2) Define the relationships between cell injury and death signaling phenotypes and damage associated molecular patterns, and immunogenic cell signaling in cancer and immune cells; (3) Validate chemical agents inducing immunogenic cell injury and death via antigen cross priming assays and deep cellular profiling using flow cytometry. Relevance: The successful completion of the proposed research is relevant to human health because it will provide methods to accelerate the identification of potential anticancer natural products, which have had and continue to have a large positive impact on human health. Furthermore, the discovery of the multiplexed cell-targeting immuno-oncological structure-activity relationships within newly discovered compounds and known but not commercially available compound families, may provide new targeted therapeutics, with greater efficiency and reduced clinical toxicity.