Molecular, Cellular, and Tissue Characterization Unit

分子、细胞和组织表征单元

• 批准号: 10900845
• 负责人: Sandro Santagata
• 金额: $ 25.44万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-04 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10900845
• 关键词: 3-Dimensional; Age; Antibodies; Antigens; Atlases; Biological Assay; Biopsy; Bone Marrow; Breslow Thickness; Cell Line; Cells; Collaborations; Collection; Competence; Core Facility; Cutaneous; DNA; DNA Damage; DNA Sequence Alteration; DNA sequencing; Data; Data Collection; Dimensions; Disadvantaged; Disease Progression; Elements; Ensure; Evaluation; Event; Fee-for-Service Plans; Fixatives; Flow Cytometry; Gene Expression; Gene Frequency; Gene Mutation; Genes; Genetic Transcription; Genotype; Health Services Research; Hematology; Heterogeneity; Hybrids; Image; Image Cytometry; Immune; Knowledge; Label; Laboratories; Lasers; Learning; Lesion; Letters; Link; Longitudinal cohort; Maintenance; Maps; Measurement; Measures; Metadata; Methods; Microscope; Mitochondria; Modality; Molecular; Morphology; Multiplexed Ion Beam Imaging; Mutation; Neoplasms; Optics; Pathologist; Patients; Phase; Play; Policies; Procedures; Prospective cohort; Proteins; Protocols documentation; Quality Control; RNA Sequences; Reagent; Reproducibility; Research Personnel; Resolution; Role; Sampling; Scanning; Series; Skin; Specimen; Stains; Standardization; Subcellular structure; System; T cell clonality; T cell receptor repertoire sequencing; Techniques; Technology; Technology Assessment; Testing; Three-Dimensional Imaging; Time; Tissue Sample; Tissues; Training; Work; cell transformation; data pipeline; experience; fluorophore; genetic variant; high dimensionality; high resolution imaging; improved; individual patient; information processing; innovation; instrument; laser capture microdissection; mRNA Expression; melanoma; member; metadata standards; multidimensional data; multiplexed imaging; mutational status; premalignant; prospective; quality assurance; reconstruction; sample fixation; single-cell RNA sequencing; transcriptome sequencing; tumor progression; ultra high resolution

CHARACTERIZATION UNIT - SUMMARY ABSTRACT The Characterization Unit will oversee collection of all image and omic data needed for construction of multi-parametric Atlases of MP lesions and CHIP specimens. Following a technology assessment/consolidation phase in year 1 most data collection will take place in the LSP and established core facilities as a means to ensure reproducibility. The Characterization Unit will work with the Analysis Unit to establish a robust information processing system that maintains the reliability of data and metadata collection and storage for Atlas specimens and QC/QA samples. Metadata will include the elements needed to comply with FAIR standards. Dissociative technologies such as single cell-RNA sequencing, DNA sequencing, and FACS/CyTOF will provide the highest dimensional data for Atlas construction but only on cells outside of their tissue context. Highly multiplexed imaging of FFPE biopsies will be used to collect spatially resolved data on cell states and morphology as well as expression of selected genes and genotypes (using padlock-rolling circle amplification probes). For the MP Atlas, laser capture of FFPE specimens will be followed by assessment of mutational status by WES, T cell clonality by TCR-seg and RNA expression by RNA-Seq. The Analysis unit will use multi-view learning and related methods to integrate the resulting spatial and omic data and to deconvolute bulk RNA sequence when scRNAseq is not possible (i.e. when analyzing FFPE MPs). Standard operating protocols developed in the Center will be evaluated and disseminated to other HTAN members. Aim 1 will characterize changes in gene expression, acquisition of mutations and cell-cell heterogeneity by sequencing, flow cytometry and CyTOF. Padlock-based FISH will be used to provide single-cell resolution of mRNA expression and mutational status. Aim 2 will collect high dimensional high resolution of images from FFPE specimens at using two complementary techniques: t-CyCIF and DEI. Antibodies will be rigorously tested and QA/QC data linked to Atlas images. A sub-set of CHIP and MP samples will be analyzed in 3D using serial sections. Aim 3 will collect time-series data from serial biopsies of CHIP patients in a longitudinal cohort and deep UMI sequencing and flow cytometry will be used to assay variant allele frequency and clonal progression. Aim 4 will implement quality assurance policies inspired by experience of quality assurance officer and pathologist Sandro Santagata in a CLIA setting. This will involve SOPs, routine retesting, competency training and statistical quality control. Aim 5 will evaluate new or improved technologies developed by PATCH Center members, other Centers in the HTAN network, collaborating investigators and companies (see letters of support). This includes direct comparison of t-CyCIF and DEI with Multiplexed Ion Beam Imaging (MIBI) now available at HMS and Scanning Mass Cytometry Imaging implemented in the Bodenmiller Lab in Zurich.

特征单元 - 摘要摘要 表征单元将监督构造所需的所有图像和毛无数据的收集 MP病变和芯片标本的多参数图谱。遵循技术 第1年的评估/合并阶段大多数数据收集将在LSP中进行并建立 核心设施作为确保可重复性的手段。表征单元将与分析合作 单元建立一个可靠的信息处理系统，该系统保持数据的可靠性和元数据的可靠性 集合标本和QC/QA样品的收集和存储。元数据将包括元素 需要遵守公平标准。解离技术，例如单细胞-RNA测序， DNA测序和FACS/CYTOF将为Atlas Construction提供最高的维数据，但 仅在组织环境之外的细胞上。 FFPE活检的高度多路复用成像将用于 收集有关细胞态和形态的空间分辨数据，以及选定基因的表达和 基因型（使用挂锁圆圈放大探针）。对于MP地图集，激光捕获FFPE 标本将通过WES评估突变状态，TCR-SEG和 RNA-seq的RNA表达。分析单元将使用多视图学习和相关方法 在Scrnaseq不是 可能（即分析FFPE MP时）。中心开发的标准操作协议将是 评估并传播给其他HTAN成员。 AIM 1将表征基因表达的变化，突变和细胞细胞的获取 通过测序，流式细胞术和细胞进行异质性。基于挂锁的鱼将用于提供 mRNA表达和突变状态的单细胞分辨率。 AIM 2将收集高维高 使用两种互补技术的FFPE标本中图像的分辨率：T-Cycif和Dei。 抗体将进行严格测试，并将QA/QC数据链接到ATLAS图像。芯片和MP的子集 将使用串行部分在3D中分析样品。 AIM 3将从串行活检中收集时间序列数据 在纵向队列和深度UMI测序和流式细胞仪中的芯片患者将用于 测定变体等位基因频率和克隆进程。 AIM 4将实施质量保证政策 受到质量保证官和病理学家桑德罗·桑塔加塔（Sandro Santagata）的经验的启发。 这将涉及SOP，常规重新测试，能力培训和统计质量控制。目标5意志 评估补丁中心成员开发的新技术或改进的技术，HTAN的其他中心 网络，合作调查人员和公司（请参阅支持信）。这包括直接比较 T-Cycif和Dei具有多路复用离子束成像（MIBI），现在在HMS和扫描质量上可用 在苏黎世的Bodenmiller实验室中实施的细胞仪成像。