We discuss our work in development of ‘Diffuse in vivo Flow Cytometry’ (DiFC) for non-invasive fluorescence enumeration of circulating tumor cells (CTCs) and describe recent progress towards human translation of DiFC. DiFC is an emerging technique wherein highly-scattered light is used to non-invasively sample blood flowing in large deep-seated blood vessels and detect fluorescently-labeled cells. The key advantages are that it allows continuous sampling of large circulating blood volumes and enumeration of rare cells over time. We discuss progress in development an application of near-infrared fluorescent molecular contrast agents for sensitive and specific labeling of CTCs directly in vivo. Candidate contrast agents include a folate receptor-targeted probe (OTL38, Cytalux), as well as new, purpose-designed pan-epithelial CTC-specific probes. We also discuss relevant tissue optics and instrumentation considerations for potential future human translation. Ultimately, DiFC could represent a new method for continuously enumerating CTCs without drawing blood samples that may enable early detection of cancer metastasis or monitoring of response to cancer therapies.
Cancer remains one of the leading causes of death worldwide despite advances in diagnostic and treatment approaches. Current methods of detection and diagnosis remain inaccessible or expensive in nature; therefore, the development of non-invasive strategies towards early-stage cancer detection are important to allow for early intervention, treatment, and access. Liquid biopsies have emerged as a non-invasive source to improve routine cancer monitoring, however cancer biomarker abundance is low, leading to limitations in detection and accuracy. The recent discovery of neoplastic circulating hybrid cells (CHCs) in peripheral patient blood provide the potential to improve detection sensitivity of blood-based assays using novel molecular-targeting contrast agents specific to both circulating tumor cells (CTCs) and CHCs. Additionally, these contrast agents can be detected using diffuse in vivo flow cytometry (DiFC), enabling non-invasive enumeration of cancer cell burden. Herein, the development and validation of near infrared (NIR) molecularly-targeted contrast agents with specificity towards epithelial biomarkers expressed on CHC and CTCs is discussed.
KEYWORDS: In vivo imaging, Near infrared, Signal to noise ratio, Flow cytometry, Luminescence, Blood, Tissues, In vitro testing, Optical filters, Fiber lasers
SignificanceDiffuse in vivo flow cytometry (DiFC) is an emerging technique for enumerating rare fluorescently labeled circulating cells noninvasively in the bloodstream. Thus far, we have reported red and blue-green versions of DiFC. Use of near-infrared (NIR) fluorescent light would in principle allow use of DiFC in deeper tissues and would be compatible with emerging NIR fluorescence molecular contrast agents.AimWe describe the design of a NIR-DiFC instrument and demonstrate its use in optical flow phantoms in vitro and in mice in vivo.ApproachWe developed an improved optical fiber probe design for efficient collection of fluorescence from individual circulating cells and efficient rejection of instrument autofluorescence. We built a NIR-DiFC instrument. We tested this with NIR fluorescent microspheres and cell lines labeled with OTL38 fluorescence contrast agent in a flow phantom model. We also tested NIR-DiFC in nude mice injected intravenously with OTL38-labeled L1210A cells.ResultsNIR-DiFC allowed detection of circulating tumor cells (CTCs) in flow phantoms with mean signal-to-noise ratios (SNRs) of 19 to 32 dB. In mice, fluorescently labeled CTCs were detectable with mean SNR of 26 dB. NIR-DiFC also exhibited orders significantly lower autofluorescence and false-alarm rates than blue-green DiFC.ConclusionsNIR-DiFC allows use of emerging NIR contrast agents. Our work could pave the way for future use of NIR-DiFC in humans.
KEYWORDS: Tissue optics, Near infrared, Monte Carlo methods, Blood vessels, Signal detection, Sensors, Optical properties, Luminescence, In vivo imaging, Signal to noise ratio
Significance: “Diffuse in vivo flow cytometry” (DiFC) is an emerging technology for fluorescence detection of rare circulating cells directly in large deep-seated blood vessels in mice. Because DiFC uses highly scattered light, in principle, it could be translated to human use. However, an open question is whether fluorescent signals from single cells would be detectable in human-scale anatomies.
Aim: Suitable blood vessels in a human wrist or forearm are at a depth of ∼2 to 4 mm. The aim of this work was to study the impact of DiFC instrument geometry and wavelength on the detected DiFC signal and on the maximum depth of detection of a moving cell.
Approach: We used Monte Carlo simulations to compute fluorescence Jacobian (sensitivity) matrices for a range of source and detector separations (SDS) and tissue optical properties over the visible and near infrared spectrum. We performed experimental measurements with three available versions of DiFC (488, 640, and 780 nm), fluorescent microspheres, and tissue mimicking optical flow phantoms. We used both computational and experimental data to estimate the maximum depth of detection at each combination of settings.
Results: For the DiFC detection problem, our analysis showed that for deep-seated blood vessels, the maximum sensitivity was obtained with NIR light (780 nm) and 3-mm SDS.
Conclusions: These results suggest that—in combination with a suitable molecularly targeted fluorescent probes—circulating cells and nanosensors could, in principle, be detectable in circulation in humans.
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