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

描述(申请人提供)：由血管生成引起的血管形态异常，即新血管的形成，是癌症肿瘤的特征，与从静止到侵袭性侵袭性转移行为的转变有关。因此，血管生成活性，如形态微血管变化所反映的，是癌症研究中评估的关键点。微血管属性的可视化和量化可以监测疾病进展和治疗反应，但目前还没有可用的样品制备方法来产生毛细血管分辨率的数据，并适用于自动化3D定量分析。到目前为止，血管显影的标本制备技术主要是为了满足个别研究的要求而开发的，并没有解决跨实验室进行标准化定量分析的需要。常规的血管指标，如密度，通常仍然依赖多个观察者从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图像数据来实现自动量化。此外，允许收集3D定量血管数据的主要技术之一--腐蚀铸造--破坏了周围组织，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改进标本制备技术，允许收集肿瘤内和支持肿瘤周围组织的高分辨率3D血管图像数据，同时允许标记相关的感兴趣分子。这些方法将进行优化，以产生能够进行自动化定量分析的血管图像数据，从而为研究与肿瘤生长和治疗相关的微血管变化引入一种更快速、更标准化的方法。适用于动物模型和固定存档组织的方法将朝着将这些技术扩展到人类活组织的最终目标发展。我们的方法将结合和优化血管生物学中使用的方案，并调整在其他领域开发的程序，例如非哺乳动物发育生物学，这些程序虽然相关，但以前从未在该领域使用过。我们建议修改这些现有的标本制备技术，并与光学透明方法相结合，以最大限度地减少与组织不透明相关的问题，生成整体标本的微血管结构的3D渲染图，同时允许标记其他感兴趣的分子。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血管；2)通过静脉注射荧光标记的凝集素来标记功能血管；3)通过抗体或核染料的整体标记来标记其他感兴趣的结构；以及4)组织透明以最大限度地减少共聚焦显微镜成像的光散射，以便从肿瘤周围和存活的肿瘤组织的深层收集数据。

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.