Sample prep methods to allow automated 3D analysis of microvessel morphology

描述（由申请人提供）：由血管生成引起的血管形态学的缺失、新血管的形成是癌性肿瘤的特征，并且与从静止到侵袭性侵袭、转移行为的转变相关。因此，由形态学微血管变化反映的血管生成活性是癌症研究中评估的关键点。微血管属性的可视化和定量允许监测疾病进展和对治疗的反应，但是目前还没有可用的样品制备方法来产生具有毛细管分辨率并且适合于自动化3D定量分析的数据。迄今为止，用于血管可视化的标本制备技术主要是为了满足个体研究的要求而开发的，尚未解决跨实验室标准化定量分析的需求。常规的血管度量，例如密度，通常仍然依赖于由多个观察者从2D石蜡切片进行耗时的手动计数，部分原因是缺乏足够质量的3D图像数据以允许自动量化。此外，允许收集3D定量血管数据的主要技术之一腐蚀铸造破坏周围组织，消除了同时探测感兴趣分子的可能性。我们的目标是创建和完善标本制备技术，允许收集肿瘤和支持肿瘤周围组织内的高分辨率3D血管图像数据，同时允许标记相关的感兴趣分子。这些方法将被优化以产生允许自动定量分析的血管图像数据，从而为与肿瘤生长和治疗相关的微血管变化的研究引入更快速、标准化的方法。将开发适用于动物模型和固定存档组织的方法，以实现将这些技术扩展到人类活检组织的最终目标。我们的方法将联合收割机和优化用于血管生物学的协议，并适应在其他领域，如非哺乳动物发育生物学，虽然相关的，以前没有在这个领域中使用的程序。我们建议修改这些现有的标本制备技术，并与光学透明方法，以尽量减少与组织不透明度相关的问题，产生3D渲染的微血管结构的整装标本，同时允许标记的其他分子的兴趣。我们的制备技术将采用以下组合：1）通过填充造影剂来铸造脉管系统; 2）通过静脉内施用的荧光标记的凝集素来标记功能性血管; 3)通过用抗体或核染料进行整体包埋标记来标记其它感兴趣的结构;以及4）组织清除以使用于通过共聚焦显微镜成像的光散射最小化，从而可以从肿瘤周围和活的肿瘤组织的深处收集数据。

DESCRIPTION (provided by applicant): Abnormalities of vascular morphology resulting from angiogenesis, the formation of new blood vessels, are characteristic of cancerous tumors and are associated with a switch from quiescent to aggressively invasive, metastatic behavior. Thus angiogenic activity, as reflected by morphological microvascular change, is a critical point of assessment in cancer research. Visualization and quantification of microvascular attributes permit monitoring of disease progression and response to therapy, yet there are no currently available sample preparation methods to produce data that are of capillary-resolution and suitable for automated 3D quantitative analysis. Specimen preparation techniques for vascular visualization thus far have primarily been developed to meet the requirements of individual studies and have not addressed the need for standardized quantitative analysis across labs. Routine vascular metrics, such as density, often still rely upon time-consuming manual counting from 2D paraffin sections by multiple observers, in part because of the lack of 3D image data of sufficient quality to allow automated quantification. Moreover, one of the principal techniques allowing the collection of 3D quantitative vascular data, corrosion casting, destroys surrounding tissue, eliminating the possibility of simultaneous probing for molecules of interest. Our goal is to create and refine specimen preparation techniques that allow collection of high-resolution 3D vascular image data within tumors and the supporting peri-tumoral tissue, while simultaneously allowing labeling of related molecules of interest. The methods will be optimized to produce vascular image data that will allow automated quantitative analysis, thus introducing a more rapid, standardized approach for studies of microvascular changes associated with tumor growth and treatment. Methods appropriate to animal models and to fixed archived tissue will be developed toward the ultimate goal of extending these techniques to human biopsy tissue. Our methods will combine and optimize protocols used in vascular biology and adapt procedures developed in other fields, such as non-mammalian developmental biology, that though relevant, have not previously been employed in this realm. We propose to modify these existing specimen preparation techniques, and in concert with optical clearing methods to minimize problems associated with tissue opacity, produce 3D renderings of the microvascular architecture of whole-mount specimens, while permitting labeling of other molecules of interest. Our preparation techniques will employ combinations of: 1) casting of the vasculature by filling with a contrast agent; 2) marking of functional blood vessels by fluorescently labeled lectin administered i.v.; 3) marking of other structures of interest by whole-mount labeling with antibodies or nuclear dyes; and 4) tissue clearing to minimize light scattering for imaging by confocal microscopy so that data can be collected from deep within peri-tumoral and viable tumoral tissue.