# Preliminary assessment of biodistribution and targeting of the fluorescent molecular probe Cy7-SYL3C in an EpCAM-positive colorectal cancer mouse model

## TL;DR

This study evaluates a fluorescent probe targeting EpCAM in colorectal cancer mice, showing it accumulates specifically in tumors and has potential for clinical imaging.

## Contribution

The study demonstrates Cy7-SYL3C's EpCAM-specific tumor targeting and metabolic pathway in mice, supporting its use as a near-infrared imaging tool.

## Key findings

- Cy7-SYL3C accumulates rapidly and specifically in HT-29 tumors, with fluorescence detected within 5 minutes of injection.
- The probe's tumor-to-muscle ratio peaked at 1.30 photons/s/mm2 six hours post-injection, showing sustained tumor retention.
- Pre-blocking with unlabeled SYL3C reduced tumor fluorescence by 55%, confirming EpCAM-specific targeting.

## Abstract

Molecular imaging probes targeting the epithelial cell adhesion molecule (EpCAM) hold considerable promise in advancing colorectal cancer (CRC) research. Building on previous work, this study further evaluated the biodistribution of Cy7-SYL3C in healthy mice and its targeting efficacy in HT-29 colorectal cancer models, confirming its potential as a near-infrared fluorescent (NIRF) imaging probe. The fluorescent molecular probe Cy7-SYL3C was synthesized by conjugating the Cy7 fluorophore to the 5’ end of the SYL3C aptamer. Biodistribution studies were conducted in healthy mice following intravenous administration of the probe. For tumor targeting evaluation, a subcutaneous HT-29 human CRC xenograft model was established in nude mice. Tumor-bearing mice were allocated into two groups: an experimental group and a pre-blocking group. The pre-blocking group received an excess of unlabeled SYL3C aptamer prior to injection of Cy7-SYL3C. Small animal in vivo imaging technology (SAFI) was employed to monitor the biological distribution and tumor targeting ability of Cy7-SYL3C at different time points from 5 min to 48 h after injection. The expression of EpCAM in tumor tissues was analyzed by Western blot. The targeting ability of the probe was evaluated through immunofluorescence co-localization and pre-blocking protocols. Cy7-SYL3C is mainly metabolized and cleared by the liver and kidneys. Fluorescence signals can be detected at the tumor site only 5 min after injection. Quantitative analysis showed that the average fluorescence intensity (AFI) at the tumor site in the experimental group was 88.2% higher (6.4 × 107 photons/s/mm2) compared to the pre-blocking group (3.4 × 107 photons/s/mm2) over a 4-hour observation period. Furthermore, the experimental group displayed a moderate positive correlation (Pearson’s r = 0.30 ± 0.02), in contrast to the negligible correlation observed in the pre-blocking group (Pearson’s r = 0.05 ± 0.01). The tumor-to-muscle ratio exceeded 1.0 at six hours post-injection and peaked at 1.30 ± 0.04 photons/s/mm2, with target-specific signals maintained for up to eight hours. This indicates that as the probe gradually removed from normal tissues such as muscles, it achieved sustained and specific retention at the tumor site. Combined with the significant reduction in the signal caused by the pre-blocking strategy, these results consistently indicated that the accumulation of Cy7-SYL3C in the body exhibited EpCAM targeting specificity. This study was based on the EpCAM targeting strategy and further evaluated the in vivo performance of the near-infrared fluorescent probe Cy7-SYL3C. The detailed dynamic imaging results at multiple time points indicated that this probe had a clear metabolic pathway in healthy mice and demonstrated rapid, sustained and specific tumor targeting ability in the HT-29 colorectal cancer model. These characteristics collectively confirmed the clinical application potential of Cy7-SYL3C as a high-performance molecular imaging tool in colorectal cancer research.

The online version contains supplementary material available at 10.1038/s41598-026-37787-2.