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Mechanistic Insights and Diagnostic Applications for Hypoxia-Induced Vasorin in Pancreatic Cancer

缺氧诱导的 Vasorin 在胰腺癌中的机制见解和诊断应用

基本信息

批准号：
10319591
负责人：
Lisa Kirkemo
金额：
$ 1.8万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2022-03-07
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10319591
关键词：
Antibodies Antigens Apoptotic Bacteriophages Binding Binding Proteins Biological Assay Biological Markers Biotinylation Cancer Cell Growth Cancer Diagnostics Cell Line Cell Proliferation Cell Survival Cell surface Cells Classification Data Data Set Detection Development Diagnosis Diagnostic Disease Disease model Early Diagnosis Early Intervention Environmental Risk Factor Enzyme-Linked Immunosorbent Assay Epitopes Event Exhibits Fab Immunoglobulins Fibroblasts Future Genes Glioblastoma Goals Growth Hematologic Neoplasms Human Hypoxia Image Immune ImmunoPET Immunoprecipitation In Vitro Label Link Malignant Neoplasms Malignant neoplasm of pancreas Mass Spectrum Analysis Membrane Metabolic Methods Modality Modeling Molecular Target N-terminal NOTCH1 gene NU/NU Mouse Neoplasm Metastasis Non-Invasive Cancer Detection Oxidative Stress Oxygen Pancreas Pathway interactions Patients Phage Display Phenotype Play Positron-Emission Tomography Primary Neoplasm Proteins Proteolysis Proteome Proteomics Publishing Recombinant Antibody Recombinant Proteins Recombinants Role Serum Signal Transduction Site Solid Solid Neoplasm Stem Cell Development Streptavidin Stress Surface Technology Testing Therapeutic Therapeutic Intervention Tissues Toxic effect Western Blotting Zirconium antibody diagnostic antibody engineering base cancer biomarkers cancer cell cancer immunotherapy cancer stem cell cancer type cell growth clinically relevant deprivation desensitization design diagnostic tool experience experimental study glycoproteomics in vivo in vivo Model insight knock-down migration mortality mouse model novel novel strategies pancreatic cancer cells pancreatic cancer model pancreatic cancer patients pancreatic neoplasm patient prognosis programs protective effect protein expression protein protein interaction radiotracer response tool transcriptomics tumor tumor hypoxia tumor microenvironment virtual

项目摘要

Project Summary With the advent of cancer immunotherapies, patients have seen dramatic increases in the availability of treatment options for various malignancies. However, many of these modalities lack efficacy in treating solid tumors, namely because of the unique set of environmental factors that exist within these tumors. One of the most ubiquitous features within the tumor microenvironment (TME) is oxygen deprivation, also known as hypoxia. Hypoxia has long been linked to increased metastasis and poorer prognoses for patients. In response to hypoxic stress, cancer cells activate highly regulated cellular pathways and gene programs that promote survival, migration, immune privilege, and increased mortality for patients. Across all solid tumors, pancreatic cancer exhibits the most severe hypoxic phenotype, which may contribute to the high mortality rate associated with this cancer type. These hypoxia-specific downstream effects suggest that tumor hypoxia can be used to elucidate selective biomarkers of solid pancreatic cancer for diagnostic and therapeutic purposes. Here, I utilize cell surface proteomics to identify a novel hypoxia-regulated target in pancreatic cancer called vasorin (VASN), a target previously implicated in the progression of glioblastoma. Furthermore, I have shown that VASN plays an important role in the growth and survival of pancreatic cancer under hypoxic stress. Finally, I have isolated and expressed ten unique antibody clones against the ectodomain of VASN. Using these antibodies, I found that VASN undergoes numerous cleavage events under hypoxia in vitro, which are unique from previously published results. We hypothesize that VASN cleavage is necessary for the survival and proliferation of pancreatic cancer under hypoxia, and that classification of expression levels for cleavage-specific forms of VASN in relevant tumor models will aid the downstream development of antibody-based diagnostics for hypoxic pancreatic cancer. To test this hypothesis, I will employ proteomic characterization and recombinant protein expression to identify the in vitro cellular consequences of VASN proteolysis. In order to isolate clones towards the membrane-retained form of cleaved VASN, I will use an established phage display-based recombinant antibody strategy. A selective antibody-based biotinylation strategy will be implemented to identify novel interacting partners of the membrane- retained form of cleaved VASN that may play an important role in downstream signaling. Finally, I will functionalize my existing and future anti-VASN antibody clones for serum-based ELISA detection and radiotracer-based immunoPET imaging of VASN, respectively, which will validate the presence of proteolyzed VASN in human serum and tumors, as well as in an in vivo nu/nu mouse model of pancreatic cancer. The proposed studies will provide mechanistic insights into the functional consequences of VASN proteolysis in pancreatic cancer, as well as providing evidence for the development of non-invasive, early-detection diagnostic alternatives for a highly metastatic and deadly disease.

项目摘要随着癌症免疫疗法的出现，患者已经看到免疫疗法的可用性急剧增加。各种恶性肿瘤的治疗选择。然而，这些方式中的许多在治疗实体瘤方面缺乏功效。肿瘤，即因为这些肿瘤内存在的独特的环境因素。之一肿瘤微环境（TME）中最普遍存在的特征是缺氧，也称为缺氧。缺氧长期以来，低氧与患者转移增加和预后不良有关。响应对于低氧应激，癌细胞激活高度调节的细胞通路和基因程序，存活、迁移、免疫豁免和患者死亡率增加。在所有实体瘤中，胰腺癌症表现出最严重的缺氧表型，这可能导致与癌症相关的高死亡率。这种类型的癌症。这些缺氧特异性下游效应表明，肿瘤缺氧可用于阐明用于诊断和治疗目的的实体胰腺癌的选择性生物标志物。在这里，我利用细胞表面蛋白质组学，以确定一种新的缺氧调节靶点，在胰腺癌称为血管紧张素（Vasorin），一个先前与胶质母细胞瘤进展有关的靶点。此外，我已经证明，在缺氧应激下胰腺癌的生长和存活中起重要作用。最后，我分离出了并表达了10个针对VEGFR 2胞外域的独特抗体克隆。使用这些抗体，我发现， VEGFR 2在体外缺氧条件下经历了许多切割事件，这是以前发表的独特的结果我们假设VEGFR 2裂解对于胰腺癌的存活和增殖是必需的以及相关肿瘤中切割特异性形式VEGF 1表达水平分类模型将有助于低氧胰腺癌基于抗体的诊断的下游开发。到为了验证这一假设，我将采用蛋白质组学表征和重组蛋白表达来鉴定在体外细胞的结果，维生素C蛋白水解。为了将克隆分离到膜保留的对于切割的VEGFR 3的形式，我将使用已建立的基于噬菌体展示的重组抗体策略。选择性将实施基于抗体的生物素化策略，以鉴定膜的新型相互作用伴侣- 保留形式的切割的VEGFR 2，其可能在下游信号传导中起重要作用。最后要功能化我现有的和未来的抗VEGFR 2抗体克隆用于基于血清的ELISA检测，分别进行基于放射性示踪剂的VEGF-C免疫PET成像，这将验证蛋白水解的存在。在人血清和肿瘤中以及在胰腺癌的体内nu/nu小鼠模型中，的拟议的研究将提供机械的见解，功能的后果，钒蛋白水解，胰腺癌，以及提供证据的发展，非侵入性，早期发现诊断高转移性致命疾病的替代品