Research Project
10485123
Pancreatic adenocarcinoma is a devastating malignancy characterized by a uniquely fibrotic pancreatic tumor stroma that limits immune cell infiltration, promotes tumor growth through cancer-stromal signaling pathways and limits delivery of systemic treatments. Development of novel therapeutic strategies that target the pathophysiology of pancreatic cancer and the characteristic fibrosis associated with it are desperately needed. Recently, the process of neutrophil extracellular traps (NETs), in which activated neutrophils release their intracellular contents including DNA, histones, and granules into the extracellular tissue or circulation, have been implicated in pancreatic cancer. Protein arginine deiminase 4 (PAD4) is an enzyme that citrullinates histones to allow for unwinding and expulsion from the cell and is required for NET formation, providing a potential therapeutic target for NET inhibition in cancer. PAD4-/- mice have diminished local and systemic NET formation, resulting in limited tumor growth and improved survival in murine pancreatic cancer. In addition to promoting pancreatic tumor growth, NETs also contribute to the spread of metastatic disease and cancer-associated hypercoagulability. We have demonstrated that neutrophil DNA released from NETs also activate pancreatic stellate cells (PSCs), the principle cell responsible for fibrosis in the pancreatic tumor microenvironment (TME). Our overall objective in the current proposal is to identify the impact of NETs on hypoxia, acidosis and metabolism in the pancreatic TME, and target NETs with pharmacologic inhibitors. In Aim 1, we will utilize innovative electron paramagnetic resonance (EPR) to provide in vivo assessment of oxygen content and pH in the pancreatic TME. We will compare tumors from wild type and PAD4-/- mice, as well as mice treated with NET inhibitors to determine the influence of NETs on these parameters. NETs release damage associated pattern molecules (DAMPs), which are known to influence metabolic processes such as mitochondrial function and autophagy. In Aim 2, we will utilize the Seahorse assay to determine how NETs alter cell metabolism in primary pancreatic cancer cells, pancreatic stellate cells and whole tumors. We will also explore how NETs activate PSCs and increase fibrosis in the TME. These studies are critically important to improving our understanding of the pancreatic tumor microenvironment. This work will provide preliminary data in support of future grants funding mechanistic studies and target these processes at a time when novel therapies are desperately needed for this devastating disease.
