Regulation of tumor-associated phagocyte physiology during tumor cell clearance

项目总结/摘要 我们目前对吞噬细胞如何吞噬和消化凋亡尸体的知识仍然很少。 了解吞噬细胞吞噬和吞噬尸体的机制特别重要，因为 癌症的免疫逃避依赖于癌细胞的吞噬作用以建立致耐受性微环境。我们 在吞噬凋亡Jurkat淋巴瘤细胞的吞噬细胞中鉴定了溶质载体（SLC）蛋白网络， 具有不同的生理过程，并证明了两个SLC的功能相关性：SLC 2A 1（促进尸体 通过葡萄糖转运的摄取）和SLC 16 A1（输出抗炎分子乳酸所需）。在这 应用，我们表明，吞噬细胞调节几个不同的功能程序，以响应尸体吞噬， 消化.这些程序之一包括SLC 12 A2，其与上游激酶WNK 1/OSR 1/SPAK一起， 作为通过氯传感/通量和细胞体积调节对凋亡Jurkat细胞吞噬的生理“制动器”。这 本申请提出检验SLC 12途径的缺失/抑制将导致肺损伤增加的假设。 体外/体内腺癌清除并诱导从“致耐受性”到“免疫原性”的临床有益转变 癌细胞清除相反，SLC 12 A2缺陷型吞噬细胞主动吞噬凋亡的Jurkat细胞， 抑制典型的抗炎转录信号和诱导促炎信号 突出表现为强烈的I型干扰素应答。此外，使用靶向SLC 12 A2或 WNK 1，我们提供了靶向该途径导致体内Jurkat细胞清除率提高的原理证明。这些 这些研究将进一步深入了解一种以前未知的细胞清除调节途径， 在肺腺癌发展/进展中的作用，并探讨遗传/药物干扰 SLC 12通路的增强将促进增加的癌细胞清除和更强的抗癌免疫应答。 在指导阶段的这一应用，我将掌握实验技术的肺，如intraperitheal 注射和支气管肺泡灌洗液收集以及建立关键肺腺癌模型 本文所我将建立新的条件性缺失小鼠，以评估SLC 12通路在小鼠体内的遗传干扰。 肺腺癌的发展/进展。我们将与同事一起开发新的工具， 体内肿瘤细胞清除过程中体积调节的重要性，使我们能够了解吞噬细胞如何建立 肺腺癌发生/进展过程中的免疫耐受。在我的指导委员会的指导下， 我将加强我的科学和专业技能，为独立阶段做准备。在独立期间 在第四阶段，我将探索在肺腺癌发展/进展过程中靶向SLC 12通路，使用小 分子。此外，我将结合联合收割机在功能基因组学，T细胞生物学/抗原呈递， 凋亡细胞清除以及指导阶段开发的工具，以了解肺癌 利用吞噬细胞的体积调节来逃避免疫反应。该项目将促进我的科学和 专业培训，使我成功地建立了一个独立的学术研究计划。

Project Summary/Abstract Our current knowledge of how a phagocyte manages engulfment and digestion of apoptotic corpses remains sparse. Understanding the mechanisms by which a phagocyte engulfs and digests corpses is particularly important, because immune evasion by cancer relies on phagocytosis of cancer cells to establish a tolerogenic microenvironment. We identified a network of solute carrier (SLC) proteins in phagocytes engulfing apoptotic Jurkat lymphoma cells associated with distinct physiological processes and demonstrated functional relevance for two SLCs: SLC2A1 (facilitates corpse uptake via glucose transport) and SLC16A1 (required for export of the anti-inflammatory molecule lactate). In this application, we show that phagocytes regulate several distinct functional programs in response to corpse engulfment and digestion. One of these programs includes SLC12A2 which, together with the upstream kinases WNK1/OSR1/SPAK, act as a physiological ‘brake’ on apoptotic Jurkat cell engulfment via chloride sensing/flux and cell volume regulation. This application proposes to test the hypothesis that deletion/ inhibition of the SLC12 pathway will lead to increased lung adenocarcinoma clearance in vitro/ in vivo and induce a clinically beneficial switch from ‘tolerogenic’ to ‘immunogenic’ cancer cell clearance. Preliminarily, SLC12A2-deficient phagocytes actively engulfing apoptotic Jurkat cells exhibited suppression of the canonical anti-inflammatory transcriptional signature and induction of a pro-inflammatory signature highlighted by a robust type I interferon response. Furthermore, using bioactive small molecules targeting SLC12A2 or WNK1, we provide proof-of-principle that targeting this pathway leads to boosted Jurkat cell clearance in vivo. These studies will provide further insight into a previously unknown cell clearance regulatory pathway, the role of this pathway in lung adenocarcinoma development/ progression, and explore the hypothesis that genetic/ pharmaceutical perturbation of the SLC12 pathway will promote increased cancer cell clearance and a stronger anti-cancer immune response. During the mentored phase of this application, I will master experimental techniques for the lungs such as intratracheal injection and bronchioalveolar lavage fluid collection as well as establish crucial lung adenocarcinoma models proposed herein. I will establish new conditional deletion mice to evaluate genetic perturbation of the SLC12 pathway on the development/ progression of lung adenocarcinoma. Together with colleagues, we will develop new tools to assess the importance of volume regulation during tumor cell clearance in vivo, allowing us to understand how phagocytes establish immune tolerance during lung adenocarcinoma development/ progression. With the guidance of my mentoring committee, I will strengthen my scientific and professional skillsets in preparation for the independent phase. During the independent phase, I will explore targeting the SLC12 pathway during lung adenocarcinoma development/ progression using small molecules. Further, I will combine my areas of expertise in functional genomics, T cell biology/antigen presentation, and apoptotic cell clearance together with the tools developed during the mentored phase to understand how lung cancer exploits phagocyte volume regulation to evade the immune response. The proposed project will promote my scientific and professional training and allow me to successfully establish an independent academic research program.