Multiplexed, Quantitative Fluorescence Imaging in Tumor Sections

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 DESCRIPTION (provided by applicant): Precision medicine for cancer requires knowledge of the driver mutations in a particular patient's tumor. Recent single cell genetic studies have shown that driver mutations and cancer cell subtype are highly heterogeneous within a single patient. Thus, clinicians will need to employ experimental methods that allow observation of a variety of molecular analytes across many individual cells within a particular patient's tumor section in situ. Current tumor section analyses are not highly multiplexed and typically remain limited to ~4-5 analytes, or 7 with multi-spectral imaging. Recent technologies have pushed this number into 30s-60s, but they require expensive equipment and/or reagents, sophisticated analyses or markedly increased assay time, all of which would preclude their practical use in many clinical pathology and preclinical research laboratories. Thus, there remains a significant need for technologies that multiplex measurements in tumor sections but are widely accessible and cost-effective. We focus on addressing this need with a readily-adoptable but novel multi- spectral fluorescence-based method. It is based on the hypothesis that the power of combinatorics can be harnessed to vastly increase the number of quantifiable analytes in a mixture by permuting the wide array of available fluorophores in new ways. We term our approach combinatorial fluorescence with spectral imaging (CoFSI). CoFSI only requires the ability to perform multi-channel fluorescence excitation and emission spectral scanning, which is widely available and easy to implement in most plate/slide readers and many microscopes. Data analysis involves a straightforward, fast computational technique called linear unmixing. Preliminary simulation studies suggest that the concentrations of 123 different CoFSI probes constructed from 16 existing fluorescent proteins can be estimated simultaneously with good accuracy and precision across 3 orders of concentration magnitude with a large number of excitation channels, and 48 probes with 6 excitation channels. Similar simulation studies constrained by available tumor section imaging equipment and Alexa dyes suggest 25 simultaneous measurements are possible. Initial pilot experiments demonstrate that seven different CoFSI probe levels in a mixture can be measured both accurately and precisely using only the blue-yellow part of the spectrum. This proposal further tests the limits of CoFSI experimentally, and applies CoFSI to tumor section imaging, with two Aims: (1) Quantify the Levels of 48 Fluorescent Probes in a Mixture Simultaneously; and (2) Quantify the Spatial Distribution of 25 Analytes in Tumor Sections. If successful, CoFSI can increase quantitative fluorescence multiplexing at least ~5 to 10-fold while relying on standard lab resources and straightforward analyses. CoFSI is also feasibly compatible with other difficult-to-multiplex technologies such as high content screening, live-cell imaging, and in vivo rodent imaging, and thus may have broad impact.