# A pH-Sensitive Sprayable Fluorescent Probe Enables Accurate Visualization of Thyroid Cancer Margins for Fluorescence-Guided Surgery in Orthotopic Mouse Models

## TL;DR

A new pH-sensitive fluorescent spray helps surgeons accurately identify thyroid cancer margins in mice, improving the chances of complete tumor removal.

## Contribution

A novel pH-sensitive sprayable fluorescent probe is introduced for real-time visualization of thyroid cancer margins during surgery.

## Key findings

- The pH-sensitive probe PH10 showed selective uptake into thyroid cancer cells and low cytotoxicity.
- PH10 enabled visualization of both macroscopic tumors and microscopic positive surgical margins in orthotopic mouse models.
- Fluorescence-guided surgery using PH10 achieved complete tumor resection confirmed by histology.

## Abstract

Surgery remains the cornerstone of treatment for thyroid carcinoma. However, intraoperative identification of tumor margins remains challenging and can hinder complete tumor removal. In this study, we applied a pH-sensitive sprayable fluorescent probe to detect thyroid cancer and guide resection in orthotopic nude mouse models. The fluorophore generated bright, highly specific fluorescence signals that facilitated complete tumor resection in these models. Overall, these results highlight the potential of this pH-sensitive probe to improve surgical precision in thyroid cancer.

Positive surgical margins (PSMs) are a major predictor of recurrence in thyroid cancer; however, their intraoperative detection remains challenging, particularly for microscopic PSMs. This study aimed to demonstrate that a sprayable pH-sensitive near-infrared fluorescent probe (PH10) could specifically and robustly label thyroid tumors in orthotopic mouse models. The pH sensitivity, cytotoxicity, and cellular uptake mechanisms of PH10 were evaluated in papillary thyroid carcinoma (K1) and anaplastic thyroid carcinoma (8505C) cell lines in vitro. Orthotopic thyroid cancer mouse models were then established using both K1 and 8505C cells. In vivo fluorescence following topical spraying of PH10 was quantified after sequential washes to assess tumor-to-background ratios. Fluorescence-guided surgery (FGS) was performed to determine whether PH10 could facilitate complete resection of orthotopically grown thyroid tumors. Complete resection was validated by hematoxylin and eosin histology. PH10 demonstrated low cytotoxicity at clinically relevant concentrations in vitro and showed selective uptake into thyroid cancer cells, predominantly via organic anion transporting polypeptide-mediated transport. Topical spraying of PH10 in orthotopic thyroid cancer mouse models established from K1 and 8505C cell lines generated strong tumor-specific fluorescence. Tumor-to-background ratios were significantly higher than background and remained distinguishable after multiple washes. In both models, PH10 enabled visualization of macroscopic residual tumors and detection of microscopic PSMs, with fluorescence patterns closely corresponding to histologic findings. FGS enabled complete tumor resection, as confirmed by fluorescence and histology. In conclusion, topical application of PH10 provides rapid, tumor-specific fluorescence suitable for identifying PSMs and facilitating complete tumor resection by FGS in thyroid cancer.