Fluorescence Molecular In Vivo Liquid Biopsy of Circulating Tumor Cells

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Project Summary The goal of this project is to develop new technology to detect circulating tumor cells (CTCs) directly in the bloodstream without having to draw blood samples. Metastasis is responsible for the majority of cancer-related deaths, and is often mediated by dissemination of CTCs via the vasculature. CTCs are therefore of great interest clinically and in basic cancer research. Nearly all methods for the study of CTCs rely on drawing and analyzing small (7.5 mL) blood samples, which is broadly known as ‘liquid biopsy’. However, due to the rarity of CTCs estimation of CTC numbers from small blood samples is extremely inaccurate, and rare cells may escape detection entirely. Liquid biopsy is also insensitive to natural changes in CTC numbers that occur over short time periods. Our team recently developed new technology, Diffuse in vivo Flow Cytometry (DiFC), to detect rare, fluorescently-labeled CTCs directly in vivo in small animals. DiFC uses diffuse light to sample large circulating blood volumes in bulk tissue. The main advantage of DiFC is therefore sensitivity: we showed that DiFC can accurately detect fewer than 1 CTC per mL of PB. Because it does not require blood draws, DiFC also allows longitudinal studies in individual animals. DiFC is also readily scalable to larger limbs, species, and potentially even to humans. However, use of DiFC in humans would require bright and specific fluorescent labeling of target CTCs in vivo. Fortunately, there has been major technical and regulatory progress in injectable molecular tracer technology for fluorescence guided surgery of cancer. Of particular interest for this proposal, OTL38 is a near-infrared (NIR) small-molecule folate-receptor (FR)-targeted probe that is in phase-III clinical trials for ovarian and liver cancer. OTL38 has high specificity and affinity for CTCs in blood, with low non-specific uptake by other blood cells or vessel walls. In addition, because NIR light experiences minimal light attenuation through biological tissue, OTL38 is suitable for detection from deeper-seated blood vessels. The goal of this project is to develop and validate the enabling technology for ‘fluorescence molecular in vivo liquid biopsy of CTCs’. In Aim 1, we will build an H-DiFC system for use in the human wrist or forearm, where arterial flow rates are 100s of mL per minute. In Aim 2, we will validate labeling of FR+ CTCs with OTL38, and detectability with H-DiFC in an arm-mimicking flow phantom model in vitro. We will quantify specificity, brightness, and external detectability up to 4 mm deep in tissue. In Aim 3, we will test OTL38 and H-DiFC in a mouse metastasis model and in hairless guinea pigs (which have similar optical properties to human skin) in vivo. If successful, H-DiFC would allow sensitive and accurate enumeration of CTCs continuously without drawing blood samples. This would represent a completely new diagnostic tool for staging, managing, and studying metastasis for a broad range of malignancies. Moreover, because OTL38 (and other fluorescent tracers) are already in advanced clinical trials, there would be a rapid pathway to first-in-human testing.