Project 3

项目概述（见说明）：本研究的目的是进一步开发和应用新的工具，3D光学断层扫描和芯片实验室，以更好地了解癌细胞的物理属性。更深入地了解癌细胞的物理特性，将有助于对癌症等级、基因组和化疗干预前后进行定量比较，是治疗和预防进展的重要组成部分。这两种技术都将应用于单细胞。为了提出一个易于处理的研究计划，研究的癌细胞类型是结肠癌和食道癌，但结果可能与其他癌症有关。永生化细胞系的属性代表癌症和细胞从患者活检将被表征。光学断层扫描：3D光学断层扫描在100纳米长度范围内提供两种类型的信息：第一种是形态测量学（结构：形状，大小，染色质质地和密度），依赖于吸收成像，类似于癌症细胞病理学家几十年来用于临床诊断癌症的图像类型（H&E和Feulgen染色）。二是功能性（定位蛋白浓度），依靠抗体等荧光染色。这项工作的新贡献之一是首次使用仪器和软件进行细胞CT成像。这为悬浮在自然状态下的细胞提供了真正的各向同性分辨率，消除了由于厚切片和涂片重叠特征导致的模糊性，以及所有2D成像方法中固有的方向依赖性、平坦化导致的扭曲和不完整采样等问题。各向同性分辨率为这种方法提供了两个显著的优势：1)测量是稳健的和可重复的，因为它们代表了整个3D细胞，而不是随机选择的平面。2)测量对细胞的肿瘤进展状态非常敏感。这项研究将以前所未有的精度产生和推导核质比和倍性等参数；以及异染色质分布、粒度尺度等质地特征，以及核膜包裹、内陷、突出等指标，这些是许多癌细胞的共同特征，无法在二维上测量。芯片上的实验室：在癌症相关修饰前后，将量化细胞内和细胞间及其封闭微环境的生理参数，包括呼吸速率、pH值、离子通量和ATP浓度以及转录组水平。将探讨生理和转录组变量之间的相关性，以及这些变量与细胞CT的形态测量、密度测量、蛋白质表达水平和定位测量之间的相关性。我们的测量，以及这种相关性或缺乏相关性，在遗传和化疗修饰前后，将为癌症的生物物理学提供新的见解。

PROJECT SUMMARY (See instructions): The aim of this research is to further develop and apply the novel tools, 3D optical tomography and labs on chips, to better understand the physical attributes of cancer cells. Deeper understanding of cancer cells' physical attributes will allow their quantitative comparison between cancer grades, and before and after genomic and chemotherapeutic interventions, and is an important component of progress toward cures and preventions. Both of these technologies will be applied to single cells. To present a tractable research plan, the cancer cell types studied are colon and esophageal, but the results may be relevant to other cancers. Attributes of immortalized cell lines representing both cancers and cells from patient biopsies will be characterized. Optical Tomography: 3D optical tomography provides two types of information at the 100-nm length scale: The first, receiving primary emphasis, is morphometric (structural: shape, size, chromatin texture and density), relying on absorption imaging analogous to the type (H&E and Feulgen staining) of imagery that cancer cytopathologists have used for decades to diagnose cancer clinically. The second is functional (localized protein concentration), relying on antibody and other fluorescent staining. Among the novel contributions harnessed for this work is the instrumentation and software to perform cell CT imaging for the first time. This provides truly isotropic resolution on cells suspended in their natural state, eliminating ambiguities due to overlapping features in thick sections and smears, and problems with orientation dependence, distortions due to flattening, and the incomplete sampling inherent in all 2D imaging methods. Isotropic resolution confers two salient advantages on this method for understanding cancer: 1) Measurements are robust and repeatable, as they represent the entire 3D cell, not randomlyselected plane(s). 2) The measurements are exquisitely sensitive to the neoplastic progression status of a cell. This research will produce and derive such parameters as nuclear to cytoplasmic ratio and ploidy with unprecedented precision; and texture features like heterochromatin distribution and granularity scale, and metrics related to the nuclear membrane infoldings, invaginations and protrusions, impossible to measure in 2D, which constitute signatures common to many cancer cells. Labs on Chips: Before and after cancer relevant modifications, physiological parameters including respiration rate, pH, ion fluxes and ATP concentrations, and transcriptome levels will be quantified within and between cells and their sealed microenvironments. Correlations will be explored between physiological and transcriptomic variables, and between these and the morphometric, densitometric, and protein expression level and localization measurements from cell CT. Our measurements, and such correlations or the lack thereof, before and after genetic and chemotherapeutic modifications, will provide new insights into the biological physics of cancer.