Development and validation of technology for fluorescence detection and enumeration of extremely rare circulating cells in vivo

• Online Access: http://hdl.handle.net/2047/D20323962

Circulating tumor cells (CTCs) and CTC clusters are of great interest in studying cancer metastasis, but methods for their enumeration remain far from optimal. Current methods for enumeration of CTCs in blood rely on drawing, storing, and enriching blood samples, but these technologies may yield significant cell count errors and artifacts, and break up clusters into single cells. A promising set of technologies termed "in vivo flow cytometry" (IVFC) avoid the drawbacks of drawing blood samples, and allow continuous measurement of circulating cell populations over time rather than at discrete time points. However, microscopy-IVFC is poorly suited for detecting rare CTCs in cancer metastasis, where biologically significant numbers may be fewer than 100 cells per mL of peripheral blood. In this dissertation, we developed and performed first in vivo demonstration of a new technique called "Diffuse in vivo Flow Cytometry" (DiFC). This technology is based on a special optical fiber probe design that allows fluorescence detection of labeled circulating cells flowing in large superficial blood vessels without drawing blood samples. We first validated and characterized the fiber probe design, and showed that it significantly simplified the operation and improved signal-to-noise characteristics versus our previous work. We then developed a dual-channel DiFC design and new signal processing algorithm to analyze DiFC data. The dual-channel DiFC design allows counting of individual cells moving in arterial or venous directions, which avoids multiple counting of cells and excludes false alarms due to motion artifacts or electronic noise. We also demonstrated that DiFC allows sampling of the entire peripheral blood volume of a mouse in minutes while maintaining a false alarm rate below one per hour. To explore potential applications of DiFC, we performed a detailed characterization of DiFC capabilities. Tissue-mimicking flow phantom experiments showed that DiFC has over 95% accuracy of counting cells in the range of 0.1 to 100 detections per minute, at depths up to 2 mm in bulk scattering media, while accurately identifying the direction and speed of cells in 1-500 mm per second range. DiFC also allows detection of cells labeled with at least 105 fluorophores per cell. We then applied DiFC to non-invasively monitor CTC burden over time in a multiple myeloma disseminated xenograft model in mice. We demonstrated that the unprecedented in vivo sensitivity of DiFC allows early detection of CTC dissemination at burdens of 1 cell per mL of peripheral blood. DiFC also, for the first time in literature, detected dissemination of CTC clusters (CTCCs) in the early development of MM in vivo. We also applied DiFC for long term and non-invasive monitoring of erythrocyte camouflaged fluorescence microsensors in circulation and discussed the potential application in personalized medicine. In combination, these capabilities indicate that DiFC can be applied in a broad range application in biomedical research. We discuss future applications of DiFC and ongoing areas of technical development for DiFC.