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

North Carolina State University, Raleigh NC

Investigators

Abstract

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.