Second Harmonic Generation imaging and optical scattering probes of ovarian cancer.

建议编号：1402757P.I.：Camagnola，Paul J.标题：卵巢癌的第二次谐波成像和光学散射探针项目的意义和重要性：卵巢癌的五年存活率仍然很低(约30%)，部分原因是筛查技术不足以及对癌细胞与宿主组织相互作用的了解不足。仍然迫切需要具有足够分辨率和特异性的新技术来对显微的卵巢恶性肿瘤或前驱病变进行成像。同样重要的是要了解这些变化是如何发生的，以及它们是如何在早期和晚期疾病之间演变的。该项目将开发基于显微镜的新方法，以满足这些需求。该项目的技术说明了解卵巢癌肿瘤微环境中细胞外基质(ECM)的变化，以及这些变化如何在早期和晚期疾病之间以及不同疾病级别之间(如交界性和高度恶性)演变是很重要的。在这个项目中，我们将实施具有光学散射测量的多模式二次谐波生成(SHG)显微镜，以通过表征胶原蛋白和3D纤维结构中的大分子变化来满足这一需求。这些方法将：1)补充主要侧重于细胞的组织学，其中有不可忽略的一小部分组织被错误分类；2)根据其他患者的ECM变化提供预后信息；3)提供卵巢或输卵管的体外或最终体内成像/筛查设备的开发。SHG已经被证明是一种高度敏感和特异的成像工具，用于定量描述几种疾病中胶原组织的变化，包括许多癌症、纤维和结缔组织疾病。这一成功归功于该过程的相干性质，其中关于纤维组装的详细信息可以通过对形态、偏振特性和发射方向性的分析来获得。同样，光学散射测量已被证明是从几个器官的正常组织中区分癌症的强大工具。我们现在将利用我们之前在体外人类卵巢组织上的结果，开发新的工具来进一步了解胶原结构的变化，包括胶原大分子组装和3D纤维组织。这些都是重要的考虑因素：i)我们已经证明，正常组织和高度恶性组织之间会发生剧烈的形态变化，以及ii)据报道，几种微小的异构体在卵巢癌中表达上调，这些可能是有效的生物标志物。我们还将分别和联合使用SHG和光学散射探头询问胶原纤维构筑，并将比较不同卵巢癌类型(例如浆液性与子宫内膜型、高级别与低级别、输卵管与原发卵巢)以及原发与转移性肿瘤的重塑。测量光散射和倍频的波长依赖关系将提供不同长度尺度上的详细结构信息，并根据其结构特征划分不同的肿瘤类型。为此，将开发一种基于胶原分子变化和纤维结构变化的分类系统。这项工作的关键是开发一套新的无标记倍频偏振分析，专门探测原纤维内胶原分子排列的变化，α-螺旋螺距角度，以及胶原三螺旋的整体手性。在癌变和肿瘤生长过程中，所有这些性质都可能由于蛋白酶活性的增加或胶原异构体掺入的变化(如Col III)而改变。这些将在自组装的纤维凝胶中进行基准测试，这种凝胶模仿卵巢基质中的胶原组装，并扩展到体外人类组织。总而言之，该项目将开发探测所有水平的胶原组织的工具，并将作为卵巢癌的新的无标记生物标记物。此外，这些方法将普遍适用于其他以胶原结构变化为特征的癌症和疾病。

Proposal Number: 1402757P.I.: Campagnola, Paul J.Title: Second Harmonic Generation imaging and optical scattering probes of ovarian cancerSignificance and Importance of Project:Five year survival rates of ovarian cancer remain poor (~30%), where this arises in part due to inadequate screening technologies as well as poor understanding of how cancer cells interact with the host tissue. There remains a compelling need for new technologies that have both sufficient resolution and specificity to image microscopic malignant ovarian tumors or precursor lesions. It is also equally important to understand how these changes occur and how they evolve between early stage and advanced disease. This project will develop new microscope-based methods that address these needs. The methods to be developed will be applicable to other cancers as well.Technical Description of the ProjectIt is important to understand the changes that occur in the extracellular matrix (ECM) in the tumor microenvironment in ovarian cancer and how these evolve between early stage and advanced disease and between disease grades (e.g. borderline vs high grade). In this project, we will implement multimodal forms of Second Harmonic Generation (SHG) microscopy with optical scattering measurements to address this need by characterizing macromolecular changes in collagen and also in the 3D fibrillar architecture. The methods will i) compliment histology which primarily focuses on cells, where a non-negligible fraction of tissues are mis-classified; ii) provide prognostic information based on ECM changes from other patients; and iii) afford the development of an ex vivo or ultimately in vivo imaging/screening device of either ovaries or fallopian tubes. SHG has already been shown to be a highly sensitive and specific imaging tool for quantitatively describing changes in the collagen organization in several diseases, including many cancers, fibroses, and connective tissue disorders. This success is due to the coherent nature of the process where detailed information on fibrillar assembly can be obtained through analysis of morphology, polarization properties, and emission directionality. Similarly, optical scattering measurements have proven to be powerful for delineating cancers from normal tissue in several organs. We will now leverage our previous results on ex vivo human ovarian tissues and develop new tools to further understand changes in the collagen architecture, both in terms of collagen macromolecular assembly and 3D fibrillar organization. These are both important considerations as: i) we have demonstrated that dramatic morphological changes occur between normal and high grade malignant tissues, and ii) it has been reported that several minor isoforms become up-regulated in ovarian cancer and these may be effective biomarkers. We will also interrogate the collagen fibrillar architecture using SHG and optical scattering probes, separately and in conjunction and will compare remodeling across a spectrum of ovarian cancer types (e.g. serous vs endometroid, high grade vs low grade, fallopian tube vs primary ovary) as well as primary vs metastatic tumors. Measuring the wavelength dependences of optical scattering and SHG will provide detailed structural information across different length scales and delineate different tumor types based on their structural characteristics. To this end, a classification system based on the collagen molecular changes and fibrillar architecture alterations will be developed. Key to this work is the development of a suite of new label free SHG polarization analyses to specifically probe changes in collagen molecular alignment within fibrils, the á-helix pitch angle, and the overall chirality of the collagen triple helix. All these properties can change due to increased protease activity or changes in collagen isoform incorporation (e.g. Col III) in carcinogenesis and tumor growth. These will be benchmarked in self-assembled fibrillar gels that mimic the collagen assembly in the ovarian stroma and extended to ex vivo human tissues. In sum, this project will develop tools that probe all levels of collagen organization and will serve as new label-free biomarkers for ovarian cancer. Moreover, these methods will be generally applicable to other cancers and diseases characterized by changes in collagen architecture.