The applicants will use multiphoton excited (MPE) photochemistry to fabricate models of the ovarian extracellular matrix (ECM) as platforms to study early stages of ovarian carcinoma. The ECMs will consist of crosslinked basal lamina and stromal layers, where each will be comprised of the respective predominant protein composition in vivo. As these nano/microstructured models will simulate the crosslinked fibrillar structure of the native ECM, they will be significantly more biomimetic than those reported previously (e.g. culture coatings and flow chambers), which are limited by the lack of concurrent appropriate topographic and biochemical structure. These devices will allow the testing of pathway activity not immediately possible in human subjects or even animal models. these models will be used to investigate the cellular surface dynamics (Aim 1) and ECM remodeling of the ovarian stroma (Aim 2) that both may occur in early carcinogenesis and may underlie disease progression. To this end, they will compare the functional response of ovarian tumor cells in terms of differentiation, migration, adhesion strength, integrin expression, proliferation as well as the ability to invade and remodel the ECM. They will specifically modulate the expression of metalloproteinases (MMPs) implicated in ECM remodeling and metastasis via stimulation with cytokines and growth factors. These experiments will reveal the biochemical and morphological factors operative in carcinogenesis and metastasis.
Intellectual Merit Cancer of the ovary has the lowest survival rates of the gynecologic cancers, with a 5-year survival in the United States from 1989-1996 of 30% or less. Ovarian cancer is predominantly diagnosed in later stages because of the lack of reliable symptoms for early stage disease as well as the lack of efficacious screening techniques to reliably detect a cancer arising in the ovary prior to metastasis. There have been 4 major limitations to screening for this disease. One challenge has been the uncertainty as to whether a premalignant or precursor lesion exists. Second, the ovary is intraperitoneal and, like the pancreas, is hard to access and image. Third, the technology used for screening is nonspecific e.g. ultrasound and even the technology used for diagnostic purposes e.g. CT scan and MRI lack specificity and have many false negative and false positive readings. Lastly, and perhaps more important, is that we lack understanding of the basic science of the tumor environment. In this project we focused on developing technologies to study the cellular factors and tissue structure to determine the factors that affect tumor growth and metastasis. To this end, we developed new nano/microscale patterning technologies to make models that allow systematic hypothesis testing. A factor that has been implicated in tumor growth is the response to cells of changes in protein concentration, also known as haptotaxis. There were no known methods to create surface and 3D models to study this process. We adapted our fabrication technology to this end. We demonstrated the process using fibroblasts. We used this approach to study a series of ovarian cancer cells. In these studies, we varied the protein concentration in the patterns and studied the migration (cell movement) of a series of ovarian cancer cells of varying metastatic potential. This is important as cell migration is thought to be important in cancer growth but has historically been difficult to study with realistic tissue models. We found that more metastatic cancers cells generally migrated faster but the particulars also depended on the cell shape. This was not previously known. The largest accomplishment of the project was developing technologies to create true 3D models that were reflective of the tissue structure of the malignant ovary. Her we begin with high resolution microscopy images of the ovaries and make a model directly from these images. This is the first true imaged-based design as this size scale. This has allowed the detailed study of how cells interact with underlying tissue. An example is shown in the image. This technology has broad applicability for other cancers, and other diseases, e.g. fibroses and connective tissues disorders and cardiovascular disease, where in all these cases the details of how cells interact with the tissues is not well understood. These types of studies cannot be performed on patients and are impractical in animal models. We have also used this technology to study stem cell differentiation in 3D environments. Broader Impacts Education Several graduate students and undergraduate students and 2 postdoctoral fellows were trained during this project. The postdoctoral fellows found jobs either in industry or chose to have additional postdoctoral experience. An undergraduate is now in medical school at Boston University. The PI developed a graduate course on optical microscopy as part of the biomedical engineering curriculum. This has included a discussion of papers from the literature as well exercises in the lab. The PI also gives guest lectures in other BME courses as well as in medical physics and chemistry about imaging approaches to study cancer. Outreach For the past 3 years, The PI has been on the advisory board for the Project Lead the Way (PLTW) engineering and biomedical curricula taught at the Middleton, WI high school. PTLW is dedicated to increasing STEM participation to make our next generation students highly competitive in the global economy, where the approach consists of classroom and hands on learning, encouraging critical thinking and problem solving skills. Since the summer of 2012, one of the PTLW recent graduates (Jorge Lara) worked in my lab performing data analysis, and has continued since then and will be transferring to UW-Madison next semester. The PI is the faculty advisor for the UW-Madison student OSA and SPIE local chapters, which include all of my students. As K-12outreach, we participated every year in the Annual UW-Madison’s Science Expeditions as well as Science Saturdays at the Wisconsin Institute of Discovery, where we conducted optics demonstrations to the kids stopping by our table.
