Characterizing the biochemistry and dynamics of the immune suppressive CXCL12 coat in pancreatic cancer

胰腺癌中免疫抑制 CXCL12 外壳的生物化学和动力学特征

基本信息

批准号：
10642901
负责人：
Philip Andrew Moresco
金额：
$ 4万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-16 至 2026-07-15
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10642901
关键词：
Amino Acids Biochemical Process Biochemistry Blocking Antibodies CXC Chemokines CXCR4 Receptors Cancer Etiology Carcinoma Cell Line Cells Cessation of life Clinical Clinical Research Complex Data Detection Disease susceptibility Doxycycline Educational workshop Engraftment Enzymes Failure Fellowship Fibroblasts Future Generations Glutamine Glycocalyx Goals Growth Harvest Human Immune Immune system Immunosuppression In Vitro Keratin-19 Kinetics Knock-out Knowledge Laboratories Ligands Link Malignant Neoplasms Malignant neoplasm of pancreas Mass Spectrum Analysis Mediating Mediator Mus Mutation Pancreatic Ductal Adenocarcinoma Predisposition Procedures Process Recombinants Refractory Repression Resistance Risk Scanning Site Source Stains Stromal Cell-Derived Factor 1 Surface Survival Rate Suspensions System T-Lymphocyte Time Tissue Stains Training Tumor Immunity United States Universities Work adaptive immune response anti-PD-1 cancer cell cancer infiltrating T cells chemokine covalent bond crosslink extracellular fibroblast activation protein alpha functional loss immune activation immune checkpoint blockade in vivo insight keratin 12 laboratory experience matrigel meetings mouse model mutant pancreatic ductal adenocarcinoma model pre-clinical preclinical study prevent receptor response subcutaneous transcriptome sequencing transglutaminase 2 tumor tumor growth tumor immunology vector

项目摘要

Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer related deaths in the United States, with a five-year survival rate of 7-8%. This poor survivability can be partially attributed to late detection, however PDAC therapies have remained stagnant which amplifies the discordance between the survivability of PDAC and other cancers. Immune checkpoint blockade (ICB) has revolutionized the treatment of certain malig- nancies, but most carcinomas, including PDAC, remain refractory. The Fearon Lab has identified that the chem- okine (C-X-C motif) ligand 12 (CXCL12) “coats” cancer cells in mouse models of PDAC, and that blocking the interaction between CXCL12 and chemokine (C-X-C motif) receptor 4, the CXCL12 receptor, results in T cell influx into cancer cell nests and response to ICB. Additionally, we find that CXCL12 is covalently linked to keratin 19 (KRT19) on the surface of cancer cells by the isopeptide bond forming enzyme transglutaminase 2 (TGM2), and knockout of either Krt19 or Tgm2 results in T cell entry into cancer cell nests and response to ICB. This data indicates that the CXCL12-KRT19 conjugate is a primary mediator of tumor immune suppression, however, our understanding of the biochemical process of its formation, and kinetics of its formation and disassembly, are lacking yet would provide insight for future studies attempting to regulate these processes. Therefore, I will de- termine the glutamine of KRT19 required to covalently bond to CXCL12 in vivo and determine if its mutation results in loss of CXCL12 coating and response to ICB. Additionally, I will quantify the rate of formation and disassembly of the CXCL12 coat and characterize the accompanying changes to immune cell activity. In Aim 1 I will generate the CXCL12-KRT19 conjugate using TGM2 in vitro and subject this complex to mass spectrometry analysis to identify the crosslinked residues between CXCL12 and KRT19. I will generate recom- binant KRT19 containing a Q to N mutation of the residue found to be crosslinked to CXCL12 and validate that CXCL12 can no longer be linked to KRT19. I will then express the mutant KRT19 in mouse PDA 1242 cells and validate that CXCL12 fails to become linked to cancer cells in vivo and determine if this failure results in suscep- tibility to ICB. In Aim 2 I will inject Matrigel suspensions of 1242 cells into C57BL/6J mice to facilitate early tumor harvesting and quantification of the rate of formation of the CXCL12 coat using immunofluorescent tissue stain- ing. I will use a Doxycycline-Off KRT19 expression system to study the rate of CXCL12 coat disassembly and perform immune cell profiling using RNA sequencing to determine how infiltrative T cells respond to coat disas- sembly. This proposal will study how the CXCL12 coat forms, the rate of its formation and disassembly, and highlight that modulating the CXCL12 coat reciprocally modifies anti-tumor immune activity. Additionally. this fellowship will provide training for my longitudinal goal of studying translational tumor immunology through in-lab training, workshops, meetings, and clinical work at Cold Spring Harbor Laboratory and Stony Brook University.

项目概要/摘要胰腺导管腺癌 (PDAC) 是美国癌症相关死亡的第三大原因国家的五年生存率为 7-8%，这种低生存率可部分归因于发现较晚，然而，PDAC 疗法仍然停滞不前，这放大了患者生存能力之间的不一致。 PDAC 和其他癌症的免疫检查点阻断 (ICB) 彻底改变了某些恶性肿瘤的治疗。南希，但大多数癌症，包括 PDAC，仍然难以治愈。 okine（C-X-C 基序）配体 12 (CXCL12) 在 PDAC 小鼠模型中“包裹”癌细胞，并阻断 CXCL12 和趋化因子（C-X-C 基序）受体 4（CXCL12 受体）之间的相互作用导致 T 细胞此外，我们发现 CXCL12 与角蛋白共价连接。 19 (KRT19) 通过异肽键形成酶转谷氨酰胺酶 2 (TGM2) 作用于癌细胞表面， Krt19 或 Tgm2 的敲除会导致 T 细胞进入癌细胞巢并对 ICB 做出反应。表明 CXCL12-KRT19 缀合物是肿瘤免疫抑制的主要介质，然而，我们的了解其形成的生化过程及其形成和分解的动力学缺乏将为未来尝试调节这些过程的研究提供见解。终止体内与CXCL12共价结合所需的KRT19的谷氨酰胺并确定其突变是否另外，会导致 CXCL12 涂层的损失和对 ICB 的反应，我将量化形成速率和 CXCL12 外壳的分解并表征免疫细胞活性的伴随变化。在目标 1 中，我将使用 TGM2 在体外生成 CXCL12-KRT19 缀合物，并对该复合物进行质量分析我将通过光谱分析来识别 CXCL12 和 KRT19 之间的交联残基。 binant KRT19 含有发现与 CXCL12 交联的残基的 Q 到 N 突变，并验证 CXCL12 不再能够与 KRT19 连接，然后我将在小鼠 PDA 1242 细胞中表达突变型 KRT19，并且验证 CXCL12 未能在体内与癌细胞相关，并确定这种失败是否会导致成功在目标 2 中，我将向 C57BL/6J 小鼠注射 1242 细胞的基质胶悬浮液以促进早期肿瘤的形成。使用免疫荧光组织染色收获和定量 CXCL12 涂层的形成速率我将使用 Doxycycline-Off KRT19 表达系统来研究 CXCL12 外壳的分解率和使用 RNA 测序进行免疫细胞分析，以确定浸润性 T 细胞如何应对外壳疾病该提案将研究 CXCL12 涂层的形成方式、其形成和分解的速率以及强调调节 CXCL12 外壳可以相互改变抗肿瘤免疫活性。奖学金将为我通过实验室研究转化肿瘤免疫学的纵向目标提供培训在冷泉港实验室和石溪大学进行培训、研讨会、会议和临床工作。