Collaborative Research: Tools for Noninvasive Nano-Optical Imaging of the Role of Extracellular Matrix in Pre-Malignant Breast Cancer

尽管进行了广泛的研究，但导致正常细胞变成癌前细胞的因素仍然未知。该项目支持研究新的成像方法，以更好地了解癌前细胞是如何被局部环境中的组织纤维（称为细胞外基质（ECM））和来自周围细胞的信号所塑造的。有证据表明，某些类型的乳腺癌，称为基底样乳腺癌（BBCs），在ECM纳米结构变化的反应中进展得更快。在三维（3D）的类组织环境中研究癌前BBCs，以模拟真实组织的许多特征。然而，这种厚的3D模型很难用传统的显微镜来测量，而且那些太小而不能用显微镜看到的特征信息也不能在不杀死细胞的情况下被确定。这项研究通过开发在ECM内部移动的纳米颗粒来解决这些限制，并且可以用外部磁铁牵引。一种使用光波的非侵入性成像技术，被称为光学相干断层扫描（OCT），将被优化并用于跟踪纳米颗粒运动和确定ECM孔径、纤维排列和刚度。研究将在3D模型中进行，以揭示在BBC的早期阶段ECM是如何重组的。此外，该研究将回答在邻近支持细胞或某些与BBC进展相关的生长因子存在时，重塑是否发生不同的问题。对BBC形成的根本性新见解可能对人类健康产生广泛影响。新的成像工具和新型光磁纳米颗粒的开发将广泛用于研究其他3D器官组织模型，这些模型越来越引起人们的兴趣，因为它们减少了动物的使用，同时为科学研究提供了一个可控的平台。计划开展教育和推广活动，让初高中学生和教师通过讲座和实践活动来学习光学物理和纳米技术。外展活动的另一个目标是促进学生的专业发展，包括提高他们对在STEM领域攻读本科学位的机会的认识。该项目通过使用等离子体金纳米棒（gnr），解决了对早期乳腺癌3D体外模型中ECM纳米结构和刚度特性的无创评估工具的需求，该工具易于扩散到3D培养物中，并与OCT相结合，提供深度分辨率成像。这项研究有三个目标。第一个目标是开发和验证GNR的扩散张量OCT用于测量各向异性基质孔径，首先是构建光学扫描仪，用OCT检测角度相关的GNR扩散，并开发并行扫描方法，以获得每个样本体素6个独特的角度测量值。将合成不同尺寸的gnr，以探索扩大孔径敏感范围。这将是第一个能够在~10微米的空间分辨率尺度上量化粒子扩散张量的仪器。第二个目标是开发和验证用于空间分辨矩阵刚度的磁性gnr，首先是开发一种使用磁性gnr （mag - gnr）结合磁动机OCT （MM-OCT）进行实时、空间分辨ECM刚度测量的技术。所开发的方法有望提供与胶原基质中的杨氏模量成正比的刚度参数。这将是第一个磁动势方法，可以解开粒子密度和刚度之间的耦合，通过使用新型磁磁- gnr。第三个目标是在BBC的三维器官型模型中量化正常到恶性前发展过程中的纳米级ECM特性。利用目标1和目标2下开发的技术，将测量从正常乳腺到导管原位癌（DCIS）进展过程中的ECM纳米结构、排列和刚度。乳腺上皮细胞类器官将与间质成纤维细胞共培养，以概括间质上皮细胞信号传导。这些技术将用于消除孔隙大小和刚度对乳腺上皮细胞类器官行为的影响。具体来说，由于恶性前病变的特征是成纤维细胞增殖增加，ECM纤维密度和排列增加，因此早期微环境变化将通过从正常到dcis样的基底样乳腺细胞等基因系进行研究。这些实验有望促进对从正常到恶性前病变过程中BBCs与微环境之间独特相互作用的理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Despite extensive research, much is still unknown about factors that cause normal cells to become pre-cancerous cells. This project supports research into novel imaging methods to better understand how pre-cancerous cells are shaped by tissue fibers in their local environment, termed the extracellular matrix (ECM), and signals from surrounding cells. There is evidence that certain types of breast cancers, called basal-like breast cancers (BBCs,) progress more rapidly in response to changes in ECM nanostructure. Pre-cancerous BBCs are studied in a three-dimensional (3D), tissue-like environment to model many features of real tissues. The thick, 3D models are difficult to measure with traditional microscopes, however, and information about features that are too small to be seen with a microscope cannot be determined without killing the cells. This research addresses these limitations by developing nanoparticles that move inside the ECM and can be pulled with an external magnet. A non-invasive imaging technique that uses light waves, known as Optical coherence tomography (OCT), will be optimized and used to track nanoparticle motions and determine ECM pore size, fiber alignment, and stiffness. Studies will be performed in 3D models to reveal how the ECM is restructured in the very early stages of BBC. In addition, the research will answer the question of whether remodeling occurs differently in the presence of adjacent supporting cells or certain growth factors implicated in BBC progression. Fundamental new insights into BBC formation have potentially broad impacts on human health. The development of new imaging tools and novel optical-magnetic nanoparticles will be broadly useful for studying other 3D organ tissue models, which are of increasing interest as they reduce the use of animals while providing a controlled platform for scientific studies. Education and outreach activities are planned to engage middle and high school students and teachers in learning about optical physics and nanotechnology through lectures and hands-on activities. Another goal of the outreach activities is to foster the students' professional development, including raising their awareness about opportunities to pursue undergraduate degrees in STEM fields.This project addresses the need for tools to noninvasively assess ECM nanostructure and stiffness properties within 3D in vitro models of early stage breast cancer by employing plasmonic gold nanorods (GNRs) that readily diffuse into and access 3D cultures, in combination with OCT to provide depth-resolved imaging. The research is organized under three objectives. The first objective is to develop and validate diffusion tensor OCT of GNRs for measuring anisotropic matrix pore sizes, beginning with construction of an optical scanner to sense angle-dependent GNR diffusion with OCT and developing methods for parallelized scanning to obtain 6 unique angle measurements per sample voxel. GNRs of varying sizes will be synthesized to explore extending the pore size sensitivity range. This will be the first instrument capable of quantifying the diffusion tensor of particles over a spatial resolution scale of ~10 micrometers. The second objective is to develop and validate Magnetic GNRs for spatially-resolved matrix stiffness, beginning with development of a technology for real-time, spatially-resolved ECM stiffness measurement using magnetic GNRs (Mag-GNRs) in combination with magnetomotive OCT (MM-OCT). The method developed is expected to provide a stiffness parameter that is proportional to Young's modulus in collagen matrices. This will be the first magnetomotive method that can disentangle the coupling between particle density and stiffness by use of the novel Mag-GNRs. The third objective is to quantify nanoscale ECM properties during normal-to-pre-malignant progression in a 3D organotypic model of BBC. Using technologies developed under objectives 1 and 2, ECM nanostructure, alignment, and stiffness will be measured during the progression from normal breast to ductal carcinoma in situ (DCIS). Mammary epithelial cell organoids will be cocultured with stromal fibroblasts to recapitulate stromal-epithelial cell signaling. These techniques will be used to dis-ambiguate the effects that pore size and stiffness each confer on the behavior of mammary epithelial cell organoids. Specifically, because pre-malignant disease is characterized by increased fibroblast proliferation and increased density and alignment of ECM fibers, early-stage microenvironmental changes will be studied with an isogenic line of basal-like mammary cells that range from normal to DCIS-like. These experiments are expected to advance understanding of the distinct interactions that BBCs exhibit with the microenvironment during the progression from normal to pre-malignant disease.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.