Impact of Neutrophil Extracellular Traps (NETs) on the Pancreatic Tumor Microenvironment

胰腺癌是一种破坏性的恶性肿瘤，其特征是一种独特的纤维性胰腺癌。 肿瘤间质限制免疫细胞渗透，通过癌症间质信号促进肿瘤生长 系统性治疗的途径和限制。开发针对以下目标的新治疗策略 胰腺癌的病理生理和与其相关的特征性纤维化是非常严重的。 需要的。最近，中性粒细胞胞外陷阱(NETs)的过程，激活了中性粒细胞的释放 它们的细胞内内容物包括DNA、组蛋白和进入细胞外组织或循环的颗粒， 与胰腺癌有牵连。蛋白质精氨酸脱亚胺酶4(PAD4)是一种 瓜氨酸组蛋白允许解离和从细胞中排出，是网状形成所必需的， 为癌症的净抑制提供潜在的治疗靶点。PAD4-/-小鼠的局部和 全身净形成，导致小鼠胰腺癌有限的肿瘤生长和提高的存活率。 除了促进胰腺肿瘤的生长，NETs还有助于转移疾病的传播 和癌症相关的高凝状态。我们已经证明了从Net释放的中性粒细胞DNA 也激活胰腺星状细胞(PSCs)，这是导致胰腺肿瘤纤维化的主要细胞 微环境(TME)。在目前的建议中，我们的总体目标是确定母语英语教师对 胰腺TME中的缺氧、酸中毒和代谢，以及药物抑制剂的靶向Net。在……里面 目的1，我们将利用创新的电子顺磁共振(EPR)来提供体内评估 胰腺TME中的氧含量和pH。我们将比较来自野生型和PAD4-/-小鼠的肿瘤，如 以及用Net抑制剂治疗的小鼠，以确定Net对这些参数的影响。篮网 释放损伤相关的模式分子(DAMP)，它们已知会影响新陈代谢过程 例如线粒体功能和自噬。在目标2中，我们将利用海马氏试验来确定 Net如何改变原发胰腺癌细胞、胰腺星状细胞和整个肿瘤中的细胞代谢。 我们还将探索Net如何激活PSCs并增加TME中的纤维化。这些研究是至关重要的 对提高我们对胰腺肿瘤微环境的了解很重要。这项工作将提供 支持未来赠款资助机制研究的初步数据，并同时针对这些过程 当迫切需要新的疗法来治疗这种毁灭性的疾病时。

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.