A dual-modality quantitative phase and polarized light microscope to assess cell motility and extracellular matrix remodeling during invasion

双模态定量相和偏光显微镜评估侵袭过程中的细胞运动和细胞外基质重塑

• 批准号: 9924599
• 负责人: Christopher B Raub
• 金额: $ 7.95万
• 依托单位: CATHOLIC UNIVERSITY OF AMERICA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9924599
• 关键词: 3-Dimensional; Actins; Biological Assay; Biopsy; Birefringence; Breast Cancer cell line; Caliber; Calibration; Cancer Biology; Cancer Model; Cancer cell line; Cells; Cessation of life; Characteristics; Clinical; Collagen; Complex; Confocal Microscopy; Development; Disease; Dose-Limiting; Effectiveness; Environment; Extracellular Matrix; F-Actin; Fibrosis; Future; Goals; Health; Holography; Hour; Hydrogels; Image; Immunofluorescence Immunologic; In Vitro; Interstitial Collagenase; Invaded; Knowledge; Label; Laser Scanning Microscopy; Length; Light Microscope; Link; MDA MB 231; Malignant Neoplasms; Maps; Measurement; Microscope; Microscopy; Microspheres; Modality; Modeling; Neoplasm Metastasis; Neural Tube Development; Optics; Pathologic Processes; Phase; Phenotype; Phototoxicity; Polarization Microscopy; Polystyrenes; Process; Proteins; Publishing; Research; Resources; Sampling; Signal Transduction; Slide; Solid; Specimen; Stains; Stress; System; Testing; Time; Tissues; Toxic effect; Wound models; base; cancer cell; cancer therapy; cell motility; cellular imaging; digital; drug candidate; flexibility; gastrulation; imaging biomarker; imaging modality; in vitro Model; indexing; inhibitor/antagonist; innovation; insight; instrument; microscopic imaging; non-invasive imaging; novel; polarized light; prognostic value; reconstruction; scale up; success; targeted treatment; therapeutic candidate; three-dimensional modeling; tumor microenvironment

7. Project Summary/Abstract Cancer metastases are responsible for most deaths from the disease. However, most current cancer therapies are anti-proliferative, rather than anti-metastatic. Challenges to the clinical realization of anti-metastatic therapies include dose-limiting toxicity to non-cancer cells, effective targeting, effective timing of administration given that metastasis is an early cancer process, and effectiveness in the face of adaptive invasion strategies by cancer cells. An ideal platform to test anti-metastatic therapeutic candidates would mimic the tumor microenvironment, and accurately assess cancer cell motile phenotypes as well as pro-invasion microstructural signatures in the extracellular matrix. This proposal aims to 1) develop a dual-modality quantitative phase and polarized light microscopy system capable of 2) evaluating the effects of inhibitors of matrix invasion and microenvironmental factors on the spread of cancer cells in a tissue-like in vitro environment. Quantitative optical indices from the proposed system accurately assess cell phenotype and microstructural signatures of invasion, by evaluating cell phase and matrix birefringence signals. These imaging modalities also deliver low optical power to the sample, allowing for long-term, serial microscopy without phototoxic effects on cancer cell movement. Finally, an innovative but simple culture set-up creates collagen networks with alignment and pre-stress similar to the tumor microenvironment. Steps to achieve these aims include addition of polarizing optics to an existing digital holographic microscope, signal calibration, channel co-registration, and initial time-lapse imaging of an in vitro 3D model of cancer invasion. Optical indices of invasion will be evaluated in a scaled-up study. After installing a second camera and full polarization state generator and analyzer on the existing digital holographic microscope, phase and birefringence signals will be evaluated and co-registered using a polystyrene microsphere standard (n=1.59) set in solid mounting media (n=1.52). Phase and polarized light parameters will be calibrated by computing phase maps of standard beads of fixed diameter, and optical retardance of a zero-order waveplate. The invasion of the breast cancer cell line MDA-MB-231 will be evaluated from serial time-lapse imaging over 24 hours, in the presence and absence of 30 nM chondramide, an actin-stabilizing anti-metastatic therapeutic candidate. The effects of pre-stress and extracellular matrix alignment will also be evaluated. The health-relatedness of this proposal lies in development of a quantitative phase and polarized light microscope that computes parameter maps for invading cancer cells and their microenvironmental surroundings. The proposed microscope reduces phototoxicity and provides quantitative metrics for accurate assessment of the mechanisms and aggressiveness of cancer cell invasion, thus enabling testing of anti- metastatic drug candidates.

7.项目总结/摘要 癌症转移是导致大多数死亡的原因。然而，目前大多数癌症疗法 是抗增殖的，而不是抗转移的。抗转移药物临床应用面临的挑战 治疗包括对非癌细胞的剂量限制性毒性、有效的靶向、有效的给药时机 考虑到转移是一个早期癌症过程， 被癌细胞杀死一个理想的平台，以测试抗转移治疗的候选人将模拟肿瘤 微环境，并准确评估癌细胞运动表型以及促侵袭微结构 细胞外基质中的信号。 本计画的目的在于：1）发展一套双模态定量相位与偏光显微镜系统 能够2）评估基质侵入抑制剂和微环境因素对 癌细胞在组织样体外环境中扩散。从建议的定量光学指数 通过评估细胞时相，系统准确地评估细胞表型和侵袭微结构特征 和矩阵双折射信号。这些成像模式还向样品传递低光功率， 允许长期、连续的显微镜检查，而对癌细胞运动没有光毒性作用。最后通过一个 创新但简单的培养设置创建了具有对齐和预应力的胶原蛋白网络， 肿瘤微环境实现这些目标的步骤包括将偏振光学器件添加到现有的数字光学器件中。 全息显微镜、信号校准、通道配准和体外细胞初始延时成像 癌症侵袭的3D模型。将在扩大研究中评价侵袭的光学指数。 在现有的数码相机上安装第二台相机和全偏振态发生器和分析仪后， 全息显微镜，相位和双折射信号将被评估和共同注册使用 聚苯乙烯微球标准品（n=1.59）置于固体封固介质（n=1.52）中。相位和偏振光 参数将通过计算固定直径的标准珠的相位图来校准， 零阶波片的延迟。乳腺癌细胞系MDA-MB-231的侵袭将是 在存在和不存在30 nM chondramide的情况下，从24小时内的连续延时成像评价， 一种肌动蛋白稳定的抗转移治疗候选物。预应力和细胞外基质的作用 还将评估对齐情况。 该建议的健康相关性在于发展定量相位和偏振光 显微镜，计算入侵癌细胞及其微环境的参数图 四周所提出的显微镜减少了光毒性，并提供了定量指标，以准确 评估癌细胞侵袭的机制和侵袭性，从而能够测试抗- 转移性候选药物