Elucidating cancer-intrinsic mechanisms of perineural invasion in pancreatic cancer

阐明胰腺癌神经周围浸润的癌症内在机制

• 批准号: 10428889
• 负责人: William L Hwang
• 金额: $ 31.51万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10428889
• 关键词: Address; Adrenergic beta-Antagonists; Advisory Committees; Alleles; Area; Biochemical; CRISPR-mediated transcriptional activation; Cancer Biology; Cancer Etiology; Candidate Disease Gene; Cell Nucleus; Cells; Cessation of life; Coculture Techniques; Collaborations; Committee Members; Critical Pathways; Custom; Data; Development; Development Plans; Epithelial; Excision; Fibroblasts; Formalin; Frequencies; Future; Gene Expression; Gene set enrichment analysis; Genes; Genetic Heterogeneity; Genetically Engineered Mouse; Goals; Guide RNA; Heat-Shock Response; Human; Immune; Immunofluorescence Immunologic; Immunohistochemistry; Invaded; Investigation; Kinetics; Knock-in; Knowledge; Libraries; Link; Literature; Machine Learning; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Maps; Mediating; Mediator of activation protein; Mentors; Mesenchymal; Metabolic; Molecular; Morbidity - disease rate; Nature; Neoplasm Metastasis; Nerve; Nerve Fibers; Neuroglia; Neurons; Non-Malignant; Organoids; Outcome; Pain; Pancreas Transplantation; Pancreatic Ductal Adenocarcinoma; Paraffin Embedding; Pathologic; Pathologic Processes; Patients; Peripheral Nerves; Phenotype; Physicians; Play; Principal Investigator; Prognosis; Property; Proteomics; Recruitment Activity; Recurrence; Research; Research Proposals; Resolution; Role; SEMA3E gene; Sampling; Scientist; Small Nuclear RNA; Synaptic plasticity; Techniques; Tissue Microarray; Tissue-Specific Gene Expression; Tumor Biology; United States; Universities; Work; axon guidance; base; cancer cell; cancer initiation; career development; cell type; cohort; density; digital; epidemiology study; experience; glial cell-line derived neurotrophic factor; human data; immunoregulation; improved; in vivo; in vivo Model; live cell imaging; lymphatic Invasion; machine learning classifier; medical schools; mortality; mouse model; multiplexed imaging; nano-string; neoplastic; neoplastic cell; neurogenesis; neuronal tumor; neurotransmission; novel; organoid transplantation; perineural; preclinical study; preservation; programs; recruit; spatial relationship; symposium; targeted treatment; therapeutic development; therapy development; therapy resistant; transcriptome; transcriptome sequencing; transcriptomics; transplant model; tumor; tumor microenvironment; tumorigenesis; tumorigenic

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer death in the United States by 2030. One of the differentiating hallmarks of PDAC is an exceptionally high frequency of perineural invasion (PNI), a histopathologic manifestation of tumor-nerve crosstalk whereby cancer cells recruit, migrate towards and invade peripheral nerves. These tumor-nerve interactions may be biochemical, electrical, and metabolic in nature and involve both neoplastic and non-neoplastic cells. While it has become apparent that intra-tumoral nerves play an important role in cancer initiation, progression, recurrence, treatment resistance, metastasis, and survival, the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown. To address this gap in knowledge, this proposal aims to perform a comprehensive spatially-resolved whole transcriptome screen in human PDAC to identify cell-type specific genes linked to PNI and dissect their functional roles by applying CRISPR activation (CRISPRa) in a novel orthotopic organoid transplant model. It is anticipated that this work will uncover a set of previously unknown mediators of PNI and provide a high-resolution molecular interconnectivity map of the tumor-nerve interactome, which will guide therapeutic development. Prior work studying molecular mediators of PNI using bulk or single-cell/nucleus RNA-sequencing lacked spatial context. The first aim applies a transcriptomic and proteomic digital spatial profiling (DSP; Nanostring) approach optimized for formalin-fixed paraffin-embedded sections of human PDAC to preserve the spatial relationships among different cell types/states. By performing DSP on tissue microarrays created from a custom cohort of PDAC patients with tumors spatially annotated for regions with and without PNI, the ability to extract gene expression differences that associate with PNI isolated from inter-patient genetic heterogeneity is greatly enhanced. The second aim will investigate the potential causal relationships between a shortlist of ~20 candidate genes and the development of PNI by performing an arrayed in vivo CRISPRa screen involving orthotopically transplanting pancreatic cancer organoids expressing single-guide RNAs (sgRNAs) targeting one or more candidate genes (KrasLSL-G12D/+;Trp53FL/FL;Rosa26LSL-dCas9-VPR-mNeonGreen;sgRNA) into syngeneic recipients. The density and properties of intra-tumoral nerves/PNI will be assessed using multiplexed imaging and machine learning approaches. The long-term goal of this research is the successful development of therapeutics that disrupt adaptive tumor-nerve crosstalk in the tumor microenvironment. The research proposal is part of an extensive career development plan that includes formal educational opportunities and seminar/conference presentations to enable the principal investigator to become an independent physician-scientist in translational cancer biology. A diverse and experienced team of mentors, scientific advisory committee members, and collaborators at MIT, MGH, DFCI, HMS, and Columbia University School of Medicine is committed to supporting and guiding the principal investigator toward achieving this goal.

项目摘要/摘要 胰腺导管腺癌（PDAC）预计将成为癌症死亡的第二大原因 到2030年，美国。PDAC的区别标志之一是 肿瘤神经串扰的组织病理学表现（PNI），癌细胞募集， 向周围神经迁移并入侵。这些肿瘤神经相互作用可能是生化，电气的 本质上的代谢和代谢，涉及肿瘤和非塑性细胞。虽然已经显而易见 肿瘤内神经在癌症开始，进展，复发，耐药性， 转移和生存，肿瘤障碍串扰的各种分子机制在很大程度上保持不变 未知。为了解决知识的这一差距，该建议旨在执行全面的空间分辨 人类PDAC中的整个转录组屏幕，以识别与PNI相关的细胞类型特异性基因并剖析其 通过在新型的原位器官移植模型中应用CRISPR激活（CRISPRA）来实现功能作用。这是 预计这项工作将揭示一组以前未知的PNI介体，并提供高分辨率 肿瘤障碍相互作用组的分子互连图，这将指导治疗性发育。 先前的工作使用批量或单细胞/核RNA-seques-seques进行PNI的分子介质缺乏空间 语境。第一个目标应用转录组和蛋白质组学数字空间分析（DSP；纳米弦）方法 针对人类PDAC的福尔马林固定石蜡包裹的部分进行了优化，以保留空间关系 在不同的细胞类型/状态中。通过在自定义队列中创建的组织微阵列上执行DSP 有或没有PNI区域的肿瘤患者的PDAC患者，可以提取基因的能力 与从患者间遗传异质性分离的PNI相关的表达差异很大 增强。第二个目的将调查约20的入围名单之间的潜在因果关系 候选基因和PNI的开发，通过执行涉及的体内CRISPRA屏幕 针对一个靶向一个指示单个诱导RNA（SGRNA）的原始移植胰腺癌类器官针对一个 或更多候选基因（KRASLSL-G12D/+; TRP53FL/FL; ROSA26LSL-DCAS9-VPR-MNEONGREEN; SGRNA） 收件人。将使用多路复用成像评估肿瘤内神经/PNI的密度和特性 和机器学习方法。这项研究的长期目标是成功发展 在肿瘤微环境中破坏自适应肿瘤串扰的治疗剂。 研究建议是一项广泛的职业发展计划的一部分，包括正规教育 机会和研讨会/会议演讲使主要研究人员能够成为 独立的医师转化生物学生物学家。一个多元化，经验丰富的导师团队， MIT，MGH，DFCI，HMS和哥伦比亚大学的科学咨询委员会成员和合作者 医学院致力于支持和指导主要研究人员实现这一目标。