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，但关于导致正常细胞成为癌前细胞的因素仍有很多未知之处。 该项目支持研究新的成像方法，以更好地了解癌前细胞如何在其局部环境中被组织纤维（称为“细胞外基质（ECM）”）和周围细胞的信号塑造。有证据表明，某些类型的乳腺癌称为基底样乳腺癌（BBC）的进展更快，以响应ECM纳米结构的变化。在三维（3D）组织样环境中研究癌前BBC，以模拟真实的组织的许多特征。然而，传统的显微镜很难测量厚的3D模型，并且在不杀死细胞的情况下，无法确定太小而无法用显微镜看到的特征的信息。这项研究通过开发在ECM内部移动的纳米颗粒来解决这些局限性，并且可以用外部磁体拉动。 一种使用光波的非侵入性成像技术，光学相干断层扫描（OCT），将被优化并用于跟踪纳米颗粒的运动和确定ECM孔径，纤维排列和刚度。在3D模型中进行研究，以揭示ECM在BBC的早期阶段是如何重构的，以及在相邻支持细胞或BBC进展中涉及的某些生长因子的存在下，重构是否会发生不同的变化。对BBC形成的基本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，这些模型越来越受到关注，因为它们减少了动物的使用，同时为科学研究提供了可控平台。计划开展教育和外联活动，通过讲座和实践活动，让初中和高中学生和教师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，包括提高他们对攻读STEM领域本科学位的机会的认识。该项目解决了对工具的需求，通过使用容易扩散并进入3D培养物的等离子体金纳米棒（GNRs），在早期乳腺癌的3D体外模型中无创评估ECM纳米结构和刚度特性，与OCT结合以提供深度分辨成像。这项研究是根据三个目标组织的。 第一个目标是开发和验证用于测量各向异性基质孔径的GNR的扩散张量OCT，首先构建光学扫描仪以利用OCT感测角度依赖性GNR扩散，并开发并行扫描方法以获得每个样本体素的6个唯一角度测量值。将合成不同尺寸的GNR以探索扩大孔径灵敏度范围。这将是第一台能够在~10微米的空间分辨率范围内量化颗粒扩散张量的仪器。第二个目标是开发和验证用于空间分辨基质刚度的磁性GNR，首先开发使用磁性GNR（Mag-GNR）结合磁动式OCT（MM-OCT）进行实时空间分辨ECM刚度测量的技术。开发的方法预计将提供一个刚度参数，是成比例的胶原蛋白基质的杨氏模量。这将是第一个磁动力学方法，可以解开粒子密度和刚度之间的耦合，通过使用新的Mag-GNRs。 第三个目标是在BBC的3D器官型模型中量化正常至癌前进展期间的纳米级ECM特性。使用根据目标1和2开发的技术，将在从正常乳腺进展为导管原位癌（DCIS）的过程中测量ECM纳米结构、对齐和硬度。将乳腺上皮细胞类器官与基质成纤维细胞共培养，以重现基质-上皮细胞信号传导。这些技术将用于消除孔径和刚度各自对乳腺上皮细胞类器官行为的影响。具体而言，由于癌前病变的特征在于成纤维细胞增殖增加以及ECM纤维的密度和排列增加，因此将使用范围从正常到DCIS样的基底样乳腺细胞的等基因系研究早期微环境变化。这些实验预计将促进理解的独特的相互作用，BBCs表现出与微环境从正常到癌前疾病的进展。这个奖项反映了NSF的法定使命，并已被认为是值得通过评估使用基金会的智力价值和更广泛的影响审查标准的支持。

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 "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 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, Optical coherence tomography (OCT), will be optimized and used to track nanoparticle motions and determine ECM pore size, fiber alignment, and stiffness. Studies are performed in 3D models to reveal how ECM is restructured in the very early stages of BBC, and 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.