Considerations for the use of contrast agents with diffuse in vivo flow cytometry to detect circulating cancer cell populations

Pace, J. S.; Matheson, G.; Malankar, G. S.; Wang, L.; Wong, M.; Gibbs, S. L.; Niedre, M. J.

doi:10.1101/2025.07.28.667193

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bioRxiv · 10.1101/2025.07.28.667193

Considerations for the use of contrast agents with diffuse in vivo flow cytometry to detect circulating cancer cell populations

Pace, J. S.·Matheson, G.·Malankar, G. S.·Wang, L.·Wong, M.·Gibbs, S. L.·Niedre, M. J.

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Abstract

SignificanceMetastasis is a leading cause of cancer-related deaths. Disseminated circulating tumor cells (CTCs) through the bloodstream seed metastatic tumors at distant sites. Most methods for enumerating CTCs in humans clinically rely on drawing and analyzing small blood samples, but these may yield inaccurate estimates of CTC burden and cannot measure CTC changes over time. Identification and enumeration of CTCs for experimental or clinical purposes largely rely on marker-driven analyses by flow cytometry. AimIn principle, non-invasive fluorescence enumeration of CTCs directly in vivo could provide a more accurate method for enumerating CTCs. However, this will require specific contrast agent for CTCs. The goal of this work is to define characteristics of useful CTC contrast agents and perform preliminary testing of candidate contrast agents used for fluorescence guided surgery (FGS). ApproachWe evaluated a clinical small-molecule folate receptor targeted contrast agent (OTL38, pafolacianine), a fluorogenic pan-cathepsin contrast agent (VGT-309, abenacianine), and a set of custom designed, small-molecule prostate specific membrane antigen (PSMA) targeted contrast agents. We tested these contrast agents using in vitro cell culture models and in in vivo murine models. ResultsAll tested contrast agents showed high uptake and labeling by target cell lines, but also small but significant labeling of non-cancer blood cells. Contrast agents that exhibited rapid clearance from circulation and the fluorogenic approach resulted in significantly reduced non-specific interfering background fluorescence signals. ConclusionsAlthough the fluorescence contrast agents tested have properties useful for labeling of CTCs, as yet none exhibited the required high specificity. This resulted in some labeling of non-cancer blood cells which presented false-positive CTC counts. Improved contrast agent design and multiplexed use of more than one contrast agent may improve this specificity.

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Pace, J. S., Matheson, G., Malankar, G. S., Wang, L., Wong, M., Gibbs, S. L., Niedre, M. J.. 2025-07-31. Considerations for the use of contrast agents with diffuse in vivo flow cytometry to detect circulating cancer cell populations. https://doi.org/10.1101/2025.07.28.667193

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