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

胰腺癌中免疫抑制 CXCL12 涂层的生物化学和动力学特征

• 批准号: 10450663
• 负责人: Philip Andrew Moresco
• 金额: $ 3.9万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 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/10450663
• 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

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/文摘