Niedre, M. J. | Search bioRxiv

bioRxiv2025

Engineering NIR probes to enhance affinity and clinical workflow compatibility for prostate cancer imaging

Positive surgical margins following radical prostatectomy increase the risk of biochemical recurrence and subsequent disease progression. Fluorescence guided surgery (FGS) using targeted contrast agents has shown clinical benefits for several cancer types. However, current prostate cancer targeted imaging probes exhibit long pharmacokinetic (PK) profiles, necessitating extended waiting periods or repeated hospital visits, limiting their integration into standard clinical workflow. To overcome this critical clinical compatibility challenge, we developed an innovative tri-compartment, chemistry-driven probe design strategy. Specifically, we developed a congeneric library of near infrared (NIR) water soluble fluorescent probes incorporating: (1) a glutamic acid-urea-lysine (EuK) ligand targeting prostate specific membrane antigen (PSMA); (2) a NIR heptamethine cyanine fluorophore optimized for enhanced PSMA binding via secondary binding sites interactions; and (3) distinct PK modulators residing outside the PSMA binding pocket to promote rapid off-target tissue clearance. While molecular docking scores, photophysical properties and live-cell staining results showed similar overall performance, probes bearing PK modulators produced stronger tumor-specific fluorescence in vivo than the control lacking a PK modulator. This effort enabled identification of a lead probe with robust tumor targeting and accelerated off-target clearance, providing optimal tumor-specific signal and contrast in a timeframe, fully compatible with robotic-assisted radical prostatectomy (RARP) timelines.

pharmacology and toxicology↗

bioRxiv2025

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

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.

bioengineering↗