# PSMA-targeted fluorescent probe for NIR-II imaging in prostate cancer intraoperative navigation and tumor margin mapping

## TL;DR

A new fluorescent probe for prostate cancer surgery improves tumor margin detection using near-infrared imaging and aligns with pathology results.

## Contribution

A PSMA-targeted fluorescent probe with NIR-II imaging capabilities for intraoperative navigation and histopathological margin mapping is developed.

## Key findings

- PSMA-12-IRDye800CW showed higher tumor-to-background ratios than ICG at 24 hours in xenograft models.
- Fluorescence intensity in human FFPE specimens correlated strongly with PSMA H-scores (R² = 0.8616).
- The probe demonstrated favorable biosafety with no acute toxicity or high immunogenic potential.

## Abstract

Accurate delineation of tumor margins during prostate cancer surgery remains challenging due to limited intraoperative visualization and insufficient molecular specificity. Here, we developed a PSMA-targeted near-infrared fluorescent probe, PSMA-12-IRDye800CW, that leverages the clinically used IRDye800CW scaffold and its extended emission tail beyond 1000 nm to support NIR-II fluorescence imaging for intraoperative navigation and histopathological margin mapping.

PSMA-12-IRDye800CW integrates a PSMA-targeting ligand with an albumin-binding linker to enable active targeting and circulation-assisted tumor accumulation. Optical properties, targeting specificity, imaging performance, and biosafety were evaluated in vitro, in prostate cancer xenograft models with direct comparison to indocyanine green (ICG) across defined time points, and in clinical formalin-fixed paraffin-embedded (FFPE) prostate specimens with matched histopathology and PSMA immunohistochemistry.

In 22Rv1 (PSMA⁺) xenografts, PSMA-12-IRDye800CW achieved significantly higher tumor-to-background ratios than ICG at key surgical-relevant time points, including 24 h (4.31 ± 0.17 vs. 2.65 ± 0.15), providing a practical imaging window for fluorescence-guided resection. Ex vivo tissue analyses further confirmed significantly higher fluorescence in tumors than in muscle and skin. In human FFPE specimens, fluorescence showed pathology-aligned spatial correspondence with PSMA immunohistochemistry, and fluorescence intensity correlated strongly with PSMA H-scores (R² = 0.8616, P < 0.0001), enabling micron-scale histopathological margin mapping. Multimodal biosafety assessments indicated favorable biocompatibility with no evident acute toxicity and low immunogenic potential.

PSMA-12-IRDye800CW enables NIR-II fluorescence imaging-assisted intraoperative navigation and provides a quantitative, pathology-anchored readout for histopathological margin mapping in prostate cancer, supporting further clinical validation of this PSMA-targeted strategy for fluorescence-guided surgery and margin assessment.