Multiplexed, Quantitative Fluorescence Imaging in Tumor Sections

肿瘤切片的多重定量荧光成像

基本信息

批准号：
8928922
负责人：
Marc R. Birtwistle
金额：
$ 25.81万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8928922
关键词：
Address Antibodies Antigens Architecture Area Basic Science Binding Biological Assay Biological Markers Biopsy Specimen Breast Cancer cell line Cells Chemicals Classification Clinic Clinical Data Clinical Pathology Combinatorics Computational Technique Cytometry Data Data Analyses Diagnostic Dyes Epitopes Equipment Escherichia coli Fluorescence Fluorescent Probes Genetic study Heterogeneity Image In Situ In Situ Hybridization Individual Knowledge Label Laboratories Life Malignant Neoplasms Measurement Measures Methods Microscope Molecular Mus Mutation Nucleic Acids Oncologist Patients Pharmacologic Substance Principal Investigator Proteins Reader Reagent Research Resolution Resources Rodent Sample Size Sampling Scanning Science Slide Solutions Spatial Distribution Staining method Stains System Techniques Technology Testing Time Tissues Translating Writing Xenograft procedure Yeasts base cancer cell cell type cellular imaging clinical decision-making clinically relevant combinatorial cost effective data acquisition deep sequencing drug development fluorescence imaging fluorophore imaging modality imaging probe improved in vivo insight malignant breast neoplasm novel optical spectra outcome forecast pre-clinical pre-clinical research precision medicine public health relevance research and development research study screening simulation spectrograph treatment response tumor tumor microenvironment tumor xenograft

项目摘要

DESCRIPTION (provided by applicant): Precision medicine for cancer requires knowledge of the driver mutations in a particular patient's tumor. Recent single cell genetic studies have shown that driver mutations and cancer cell subtype are highly heterogeneous within a single patient. Thus, clinicians will need to employ experimental methods that allow observation of a variety of molecular analytes across many individual cells within a particular patient's tumor section in situ. Current tumor section analyses are not highly multiplexed and typically remain limited to ~4-5 analytes, or 7 with multi-spectral imaging. Recent technologies have pushed this number into 30s-60s, but they require expensive equipment and/or reagents, sophisticated analyses or markedly increased assay time, all of which would preclude their practical use in many clinical pathology and preclinical research laboratories. Thus, there remains a significant need for technologies that multiplex measurements in tumor sections but are widely accessible and cost-effective. We focus on addressing this need with a readily-adoptable but novel multi- spectral fluorescence-based method. It is based on the hypothesis that the power of combinatorics can be harnessed to vastly increase the number of quantifiable analytes in a mixture by permuting the wide array of available fluorophores in new ways. We term our approach combinatorial fluorescence with spectral imaging (CoFSI). CoFSI only requires the ability to perform multi-channel fluorescence excitation and emission spectral scanning, which is widely available and easy to implement in most plate/slide readers and many microscopes. Data analysis involves a straightforward, fast computational technique called linear unmixing. Preliminary simulation studies suggest that the concentrations of 123 different CoFSI probes constructed from 16 existing fluorescent proteins can be estimated simultaneously with good accuracy and precision across 3 orders of concentration magnitude with a large number of excitation channels, and 48 probes with 6 excitation channels. Similar simulation studies constrained by available tumor section imaging equipment and Alexa dyes suggest 25 simultaneous measurements are possible. Initial pilot experiments demonstrate that seven different CoFSI probe levels in a mixture can be measured both accurately and precisely using only the blue-yellow part of the spectrum. This proposal further tests the limits of CoFSI experimentally, and applies CoFSI to tumor section imaging, with two Aims: (1) Quantify the Levels of 48 Fluorescent Probes in a Mixture Simultaneously; and (2) Quantify the Spatial Distribution of 25 Analytes in Tumor Sections. If successful, CoFSI can increase quantitative fluorescence multiplexing at least ~5 to 10-fold while relying on standard lab resources and straightforward analyses. CoFSI is also feasibly compatible with other difficult-to-multiplex technologies such as high content screening, live-cell imaging, and in vivo rodent imaging, and thus may have broad impact.

描述（通过应用程序提供）：癌症的精密医学需要了解特定患者肿瘤中的驱动器突变。最近的单细胞遗传研究表明，单个患者的驱动突变和癌细胞亚型高度异质。这是临床医生需要采用实验方法，以便在特定患者的肿瘤部分原位观察许多分子分析物。当前的肿瘤截面分析不是高度多重的，通常仍限于〜4-5个分析物，或具有多光谱成像的7个分析物。最近的技术将这个数字推向了30-60S，但是它们需要昂贵的设备和/或试剂，复杂的分析或显着增加了测定时间，所有这些都将排除它们在许多临床病理学和临床前研究实验室中的实际使用。这仍然是对肿瘤切片中多重测量的技术的重要需求，但易于获取且具有成本效益。我们专注于通过易于预先添加但新颖的基于荧光的方法来满足这一需求。它基于以下假设：可以利用组合剂的力量通过以新的方式将多种可用的荧光团置于广泛的可用荧光团中，从而大大增加混合物中可量化的分析物的数量。我们将方法与光谱成像（COFSI）相结合。 COFSI仅需要执行多通道荧光兴奋和发射光谱扫描的能力，该扫描广泛可用且易于在大多数板/滑动读取器和许多显微镜中实现。数据分析涉及一种简单，快速的计算技术，称为线性Umbixing。初步仿真研究表明，可以在3个浓度幅度的良好准确性和精确度上轻松估算123个不同的COFSI问题的浓度，并在3个浓度范围内具有良好的精度，并具有大量的兴奋通道，以及48个兴奋通道的48个问题。通过可用的肿瘤部分成像设备和Alexa染料控制的类似模拟研究表明，可以进行25种简单的测量。最初的试验实验表明，仅使用光谱的蓝黄色部分，可以准确，精确地测量混合物中的七个不同的COFSI探针水平。该建议进一步测试了COFSI实验的限制，并将COFSI应用于肿瘤截面成像，其两个目的：（1）同时量化混合物中48个荧光探针的水平；（2）量化肿瘤切片中25种分析物的空间分布。如果成功，COFSI可以在依靠标准实验室资源和直接分析的同时，增加至少约5至10倍的定量荧光多路复用。 COFSI还与其他难以传播的技术兼容，例如高素质筛选，活细胞成像和体内啮齿动物成像，因此可能会产生广泛的影响。