Metastasis and resistance to therapy are the most deadly aspects of breast cancer, and yet the conditions within the tumor microenvironment that lead to cancer cell dissemination and resistance are not fully understood. The molecular events that initiate and sustain these processes occur as rare events in the tumor microenvionment and revealing these early, subtle changes in the microenvironment will require high resolution, multiparametric in vivo microscopic approaches and reporter constructs that can characterize dynamic cellular events that lead to invasion, intravasation, and colonization of distant tissues. We will monitor breast cancer cells harboring optical reporters that signal key molecular events associated with the metastatic process in culture and in two animal models using a miniature dual-axis confocal (DAC) fluorescent microscope. This multispectral microscope is capable of detecting multiple markers of disease progression, and its novel architecture has enabled radical miniaturization conducive to implantation in rodent models for continuous study of the microenvironment. Although the applications for these devices for in vivo imaging are broad, we focus here on monitoring the critical steps of epithelial to mesenchymal transition (EMT) and back-MET. EMT is a developmental process in which organized epithelial cells transition into isolated, migratory cells with mesenchymal phenotypes, and underlies key steps in the metastatic process. We have identified a novel set of alternative splicing events associated specifically with breast cancer cell EMT and MET. We propose to engineer breast epithelial cells to report these events by transducing them with switchable bichromatic fluorescent reporters that indicate EMT status based on splicing patterns. As these reporter cells initiate EMT or MET, the coupled splicing events will result in a change in fluorescent protein expression, allowing our miniature confocal microscopes to assess cellular status within the microenvironment. We will test the hypothesis that EMT splicing events associate with, and report, key early steps leading to invasion and intravasation within the breast cancer microenvironment in three aims: 1) engineer breast tumor cells to report EMT/MET-associated alternative splicing events, 2) monitor and validate EMT/MET reporter expression in labeled breast cancer cells within human breast tissue explant and ectopic mouse model systems using an existing handheld DAC microscope, and 3) adapt the DAC microscope as an implantable device to evaluate EMT/MET-associated splicing events in the tumor microenvironment of an orthotopic rat mammary carcinoma model. This work will result in the development of implantable microscopes for serial, high resolution, multiparametric imaging, and switchable molecular reporters that reveal dynamic processes within the tumor microenvironment of living animal models.
Metastasis is the process through which cancer cells depart the primary tumor to colonize and grow in distant organs, and is responsible for most breast cancer- associated mortality. The goal of our grant is to develop informative reporter constructs and miniature microscopes that can be implanted in living animal models of breast cancer and to study molecular events through dynamic imaging of individual cancer cells expressing the 'switchable' reporters that mark drug resistance and key steps in initiation of the metastatic process. This approach will allow us to identify and profile the relevant target cell populations, and verify molecular targets for the design and application of effective therapeutic interventions.
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