Molecular, Cellular, and Tissue Characterization Unit

分子、细胞和组织表征单元

• 批准号: 10005916
• 负责人: JOE W. GRAY
• 金额: $ 73.38万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-19 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10005916
• 关键词: 3-Dimensional; ATAC-seq; Aftercare; Antibodies; Antigen Presentation; Architecture; Atlases; Biopsy; CDK4 gene; Cells; Chemicals; Chromatin; Collection; Cost Control; Detection; Disseminated Malignant Neoplasm; Drug Targeting; Event; Extracellular Matrix; Face; Fluorescence; Fluorescence Microscopy; Formalin; Freezing; Gene Expression Profile; Generations; Genome; Genomic Instability; Genomics; Goals; Human; Image; Image Analysis; Immune; Immune checkpoint inhibitor; Immunofluorescence Immunologic; Immunohistochemistry; Immunomodulators; Individual; Ions; Label; Lesion; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Mechanics; Metastatic breast cancer; Molecular; Mutation; Oligonucleotides; Paraffin Embedding; Pathway interactions; Patients; Periodicity; Pharmaceutical Preparations; Plant Resins; Primary Neoplasm; Production; Radiology Specialty; Resistance; Resolution; Sampling; Scanning Electron Microscopy; Signal Transduction; Specimen; Stains; Stromal Cells; Technology; Therapeutic; Time; Tissues; Work; base; cancer cell; castration resistant prostate cancer; cohort; combinatorial; comparative; drug efficacy; epigenome; epigenomics; exhaustion; high dimensionality; hormone receptor-positive; hormone therapy; immune checkpoint; improved; indexing; individual patient; inhibitor/antagonist; insertion/deletion mutation; malignant breast neoplasm; molecular imaging; neoplastic cell; next generation; paraform; prospective; resistance mechanism; single cell sequencing; targeted treatment; technology development; triple-negative invasive breast carcinoma; tumor; tumor-immune system interactions

ABSTRACT – Characterization Unit The Characterization Unit will be responsible for omic and multiscale image analyses of pairs of biopsies taken pre- and on/post treatment from patients with metastatic breast and prostate cancer undergoing treatment with current generation pathway and immune checkpoint targeted treatments. The paired biopsies represent sensitive and resistant lesions and comparative analyses of these enable discovery of mechanisms of resistance. Twenty pairs of samples each from three treatment cohorts will be analyzed. Specifically, (a) hormone-receptor positive breast cancer (HRBC) undergoing treatment with a CDK4/6 inhibitor in combination with endocrine therapy, (b) triple negative breast cancer (TNBC) undergoing treatment with a PARP inhibitor and an immunomodulatory agent, and (c) castration resistant prostate cancer (CRPC) undergoing treatment with enzalutamide. Aim 1 will analyze OCT frozen biopsies using Topographic Single Cell Sequencing (TSCS) to cells isolated from spatially defined regions to enable analysis of genomic copy number changes, mutations, and indels and Single-cell Combinatorial Indexing ATAC-seq (sci-ATAC-seq) to elucidate chromatin accessibility in single cells. The cells analyzed using sci-ATAC-seq will be computationally mapped to cyclic immunofluorescence (cycIF) stained sec- tions based on predicted gene expression patterns. Aim 2 will analyze formalin-fixed paraffin embedded (FFPE) sections using multiplex immunohistochemistry (mIHC) and cycIF to assess the tumor and stromal cell compo- sition and the molecular states thereof. In addition, next generation cycIF using oligonucleotide labeled antibod- ies will be developed to enable high dimension cycIF staining and imaging using both conventional and super resolution fluorescence microscopy. Aim 3 will analyze paraformaldehyde fixed, resin embedded (PFRE) speci- mens using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to identify ultrastructural changes in 2D images and targeted 3D volumes with 4-nm resolution. In addition, work in this aim will assess the utility of Serial Block Face Imaging SEM (SBFI) to define 3D ultrastructural changes in larger volumes, but at lower res- olution.

摘要 – 表征单元 表征单元将负责对所采取的活检对进行组学和多尺度图像分析 接受治疗的转移性乳腺癌和前列腺癌患者的治疗前和治疗中/治疗后 当前一代途径和免疫检查点靶向治疗。配对的活检代表敏感 耐药性病变以及对这些病变的比较分析能够发现耐药机制。二十 将分析来自三个治疗组的每对样本。具体来说，（a）激素受体阳性 正在接受 CDK4/6 抑制剂联合内分泌治疗的乳腺癌 (HRBC)，(b) 正在接受 PARP 抑制剂和免疫调节剂治疗的三阴性乳腺癌 (TNBC) (c) 正在接受恩杂鲁胺治疗的去势抵抗性前列腺癌 (CRPC)。目标1将 使用拓扑单细胞测序 (TSCS) 对空间分离的细胞进行 OCT 冷冻活检分析 定义的区域能够分析基因组拷贝数变化、突变和插入缺失以及单细胞 组合索引 ATAC-seq (sci-ATAC-seq) 用于阐明单细胞中染色质的可及性。细胞 使用 sci-ATAC-seq 分析的数据将通过计算映射到循环免疫荧光 (cycIF) 染色的 sec- 基于预测的基因表达模式。目标 2 将分析福尔马林固定石蜡包埋 (FFPE) 使用多重免疫组织化学 (mIHC) 和 cycIF 切片来评估肿瘤和基质细胞成分 位置及其分子状态。此外，使用寡核苷酸标记抗体的下一代 cycIF 将开发 ies 以使用传统和超级技术实现高维 cycIF 染色和成像 分辨率荧光显微镜。目标 3 将分析多聚甲醛固定、树脂包埋 (PFRE) 的特异性 男士使用聚焦离子束扫描电子显微镜 (FIB-SEM) 来识别超微结构变化 2D 图像和目标 3D 体积，分辨率为 4 纳米。此外，这一目标的工作将评估 串行块面成像 SEM (SBFI) 用于定义较大体积但分辨率较低的 3D 超微结构变化 解决方案。