Exploring metabolic crosstalk between cancer cells and neutrophils in pancreatic ductal adenocarcinoma - PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is the most hypovascular solid tumor type, which imposes harsh nutrient and hypoxic stress on cancer cells that limits their metabolic activity. To acquire the biomass needed for survival and proliferation in this harsh tumor microenvironment (TME), pancreatic cancer cells require the support of abundant stromal cells, which include fibroblasts, macrophages, neutrophils, B/T/NK cells, mast cells, and neurons, and often comprise >50% of tumor cellularity. Specifically, cancer-associated fibroblasts in PDAC tumors have been shown to provide alanine, proline, branched chain keto-acids, monounsaturated fatty acids and glycosaminoglycans to cancer cells, all of which promote metabolic activities and fitness in the cancer cell. Macrophages and neurons have also been implicated to a lesser extent in this collaborative behavior. Still, given the rich heterocellularity and pleiotropy of essential cellular metabolic reactions, there are likely innumerable metabolite exchanges between TME-resident cells in PDAC that remain undiscovered. Positing that diffusive metabolite exchange requires cells to be physically near each other in the tumor, I performed spatial transcriptomics in 8 primary PDAC resections to nominate novel spatial cellular synapses in PDAC tumors which could be validated using experimental models. In the most hypovascular tumors, I find large aggregates of neutrophils nucleating in the lumena of classical-like but not basal-like cancer cell glands. Follow- up analysis using scRNAseq and multiplex immunofluorescence reveal that these neutrophil aggregates are neutrophil extracellular traps (NETs), formed when a neutrophil undergoes the regulated necrotic cell death process of NETosis. This finding suggested that extreme nutrient and hypoxic stress could trigger classical-like PDAC cells to secrete neutrophil chemotactic and pro-NETotic factors. Further, it is known that macropinocytic cancer cells can repurpose lipids, proteins, and sugars derived from necrotic cell debris, and since these NETs form in the spatial vicinity of cancer cells, I hypothesize that starved classical-like malignant cells recruit neutrophils and induce their NETosis, then repurpose their biomass via macropinocytosis, promoting anabolism and survival during vascular restriction. In Aim 1, I will study the first half of this starvation circuit - the changes in neutrophil behavior (chemotaxis, transepithelial migration, NETosis) that occur when they are exposed to cultures of, or conditioned media from, classical-like or basal-like cancer cells treated with tumor-physiologic levels of nutrients and oxygen. In Aim 2, I will test whether exposure to NET debris enables cancer cell anabolism and proliferation during tumor-like nutrient stress. Together, this proposed research seeks to shed light on the drivers and consequences of neutrophil infiltration and NETosis in the PDAC TME.
