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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：
10313251
负责人：
Philip Andrew Moresco
金额：
$ 3.83万
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/10313251
关键词：
Amino Acids Biochemical Process Biochemistry Blocking Antibodies CXC Chemokines CXCL12 gene CXCR4 Receptors Cancer Etiology Carcinoma Cell Line Cells Cessation of life Clinical Clinical Research Complex Data Detection Disease model Doxycycline Educational workshop Engraftment Enzymes Failure Fellowship Fibroblasts Future Generations Glutamine Goals Growth Harvest Human Immune Immune system Immunosuppression In Vitro KRT19 gene Keratin-19 Kinetics Knock-out Knowledge Laboratories Link Malignant Neoplasms Malignant neoplasm of pancreas Mass Spectrum Analysis Mediating Mediator of activation protein Mus Mutation Pancreatic Ductal Adenocarcinoma Predisposition Procedures Process Recombinants Refractory Resistance Risk Scanning Site Source Stains Stromal Cell-Derived Factor 1 Surface Survival Rate Suspensions System T-Lymphocyte Time Tissue Stains Training Tumor Immunity Tumor-infiltrating immune cells United States Universities Vision Work adaptive immune response anti-PD-1 base cancer cell chemokine covalent bond crosslink extracellular fibroblast activation protein alpha functional loss immune activation immune checkpoint blockade in vivo insight 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.

项目总结/摘要胰腺导管腺癌（PDAC）是美国癌症相关死亡的第三大原因。五年生存率为7- 8%。这种较差的生存能力可以部分归因于发现较晚，然而，PDAC治疗仍然停滞不前，这放大了患者生存能力之间的不一致性。 PDAC和其他癌症。免疫检查点阻断（ICB）彻底改变了某些恶性肿瘤的治疗，肿瘤，但大多数癌，包括PDAC，仍然难治。Fearon实验室已经确定化学- 因子（C-X-C基序）配体12（CXCL 12）“包裹”PDAC小鼠模型中的癌细胞， CXCL 12和趋化因子（C-X-C基序）受体4（CXCL 12受体）之间的相互作用导致T细胞凋亡。流入癌细胞巢和对ICB的反应。此外，我们发现CXCL 12与角蛋白共价连接， 19（KRT 19）通过异肽键形成酶转氨酶2（TGM 2）在癌细胞表面上的作用，敲除Krt 19或Tgm 2导致T细胞进入癌细胞巢并对ICB产生应答。该数据表明CXCL 12-KRT 19缀合物是肿瘤免疫抑制的主要介质，然而，我们的研究表明，了解其形成的生化过程，以及其形成和分解的动力学，然而，目前还没有的研究将为未来试图调节这些过程的研究提供见解。因此，我将- 测定KRT 19的谷氨酰胺在体内与CXCL 12共价结合所需的量，并确定其突变是否导致CXCL 12涂层的损失和对ICB的响应。此外，我将量化的形成率， CXCL 12包被的分解并表征免疫细胞活性的伴随变化。在目的1中，我将使用TGM 2在体外产生CXCL 12-KRT 19缀合物，并将该复合物进行质量分析。通过质谱分析来鉴定CXCL 12和KRT 19之间的交联残基。我会生成推荐- 发现含有与CXCL 12交联的残基的Q至N突变的二元体KRT 19，并验证 CXCL 12不能再连接到KRT 19。然后，我将在小鼠PDA 1242细胞中表达突变的KRT 19，验证CXCL 12在体内不能与癌细胞连接，并确定这种失败是否会导致suscep，对ICB的敏感性。目的2：将1242细胞的Matrigel悬浮液注射到C57 BL/6 J小鼠体内，以促进早期肿瘤的形成收获并使用免疫荧光组织染色定量CXCL 12包被的形成速率- ing.我将使用强力霉素关闭的KRT 19表达系统来研究CXCL 12外壳解体的速率，使用RNA测序进行免疫细胞分析，以确定浸润性T细胞如何对被膜损伤作出反应， sweetie。该提案将研究CXCL 12涂层如何形成，其形成和分解的速率，突出了调节CXCL 12包被抗体改变抗肿瘤免疫活性。另外。这奖学金将为我的纵向目标提供培训，通过实验室研究转化肿瘤免疫学在冷泉港实验室和斯托尼布鲁克大学参加培训、研讨会、会议和临床工作。