Ultra-Rare Cell In Vivo Flow Cytometry

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 DESCRIPTION: There are many areas of biomedical research where the study of rare circulating cells is important. Examples include cancer metastasis, hematological malignancies, organ transplant biology, immunology, and reproductive medicine and stem-cell therapies. In this project we will develop a new high-throughput optical scanner with unprecedented capabilities for studying rare circulating cells in small animals in vivo. Circulating cells are normally quantified by drawing small blood samples which are purified and analyzed with hemocytometry or flow cytometry. However, it is known that handling and purifying blood samples can affect cell viability, and that rare cells can escape detection due to the small sampling volume. More recently, `in vivo flow cytometry' (IVFC) methods have been developed that allow enumeration of cells without drawing samples. While extremely useful, these generally rely on interrogation of microscopic blood vessels with small flow rates, so that rare cells are undetectable. New high-sensitivity and high-accuracy tools for studying circulating cells are therefore greatly needed by the research community. In this proposal we will develop a miniaturized optical scanner that will use diffuse photons to interrogate circulating blood in the limb of a mouse. The technology - termed "ultra-rare cell IVFC" (UR-IVFC) - will employ a number of unique design elements including, i) multiple tomographic optical rings with fiber-coupled lasers and fluorescence detectors, ii) efficient geometric light collection, iii) frequency encoded lasers and detector channels, and, iv) advanced signal processing algorithms for accurate counting and tracking of cells. In combination, UR-IVFC will allow single-cell sensitivity in a 10 minute scan and with a false alarm rate less than 0.001 per minute. We anticipate that the unique capabilities of UR-IVFC will have immediate impact in many research fields. We will first use UR-IVFC to study treatment of multiple myeloma (MM), a hematological malignancy for which there is currently no cure. We will study "cell mobilization therapy", which is an emerging treatment strategy for MM. MM cells are chemically forced from the protective bone marrow niche into circulation where they are vulnerable to chemotherapy. We will use UR-IVFC to study whether specific clonal sub-populations of MM cells are resistant to mobilization, and we will test the efficacy of mobilizing agents for minimal residual disease (MRD). In addition to illuminating molecular mechanisms of MM biology and treatment resistance, these studies could yield new therapeutic strategies for patients suffering from MM. Moreover they are extremely difficult (or outright infeasible) to perform with existing technology.