Multiplexed, Quantitative Fluorescence Imaging in Tumor Sections

 描述(由申请人提供)：癌症的精确医学需要了解特定患者肿瘤的驱动因素突变。最近的单细胞遗传学研究表明，驱动突变和癌细胞亚型在单个患者中具有高度异质性。因此，临床医生将需要采用实验方法，允许在特定患者的肿瘤切片中原位观察跨许多单个细胞的各种分子分析物。目前的肿瘤切片分析不是高度多元化的，通常限制在~4-5个分析物，或多光谱成像的7个分析物。最近的技术将这一数字推到了30-60秒，但它们需要昂贵的设备和/或试剂、复杂的分析或显著增加的分析时间，所有这些都将阻碍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。因此，仍然需要在肿瘤切片中多路测量但可广泛使用且成本效益高的技术。我们专注于通过一种易于采用但新颖的基于多光谱荧光的方法来解决这一需求。它是基于这样一个假设，即通过以新的方式置换广泛的可用荧光团，可以利用组合学的力量来极大地增加混合物中可定量分析物的数量。我们称我们的方法为组合荧光光谱成像(CoFSI)。CoFSI只需要执行多通道荧光激发和发射光谱扫描的能力，这在大多数平板/载玻片阅读器和许多显微镜中都是广泛可用的，很容易实现。数据分析涉及一种简单、快速的计算技术，称为线性分解。初步的模拟研究表明，从16个已有的荧光蛋白构建的123个不同的CoFSI探针的浓度可以在3个数量级的浓度量级和大量的激发通道以及48个探针的6个激发通道上同时估计，并且具有良好的准确度和精密度。受现有肿瘤切片成像设备和Alexa染料限制的类似模拟研究表明，同时进行25次测量是可能的。初步的试点实验表明，只使用光谱的蓝黄部分就可以准确和精确地测量混合物中的七个不同的CoFSI探测器水平。这一建议进一步在实验上测试了CoFSI的极限，并将CoFSI应用于肿瘤切片成像，目的有两个：(1)同时定量混合物中48个荧光探针的水平；(2)定量25个分析物在肿瘤切片中的空间分布。如果成功，CoFSI可以在依赖标准实验室资源和直接分析的情况下，将定量荧光多路复用增加至少~5到10倍。CoFSI还可行地与其他难以多路复用的技术兼容，如高含量筛选、活细胞成像和活体啮齿动物成像，因此可能会产生广泛的影响。

 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.