-Objectives
Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a fundamental role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and aggressive thyroid cancer, such as anaplastic thyroid cancer (ATC). The objective of this study was to detail the synthesis and characteristics of a dual-modality imaging agent targeting PI3K, namely Tc-99m GIVAGDVTPI-HEG-ECG-K(-5-carboxy-X-rhodamine)-NH2 (GIVA-ECG-ROX). Furthermore, we aimed to verify its feasibility as a molecular imaging tool for thyroid malignancy.

-Methods
GIVA-ECG-ROX was synthesized using Fmoc solid-phase peptide synthesis. The radiolabeling of GIVA-ECG-ROX with Tc-99m was achieved through ligand exchange via tartrate. We conducted binding affinity and in vitro cellular uptake studies. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed using NCI-H460 (PI3K positive) and MNK-45 (PI3K-negative) tumor-bearing murine models.

-Results
After the radiolabeling procedures with Tc-99m, Tc-99m GIVA-ECG-ROX complexes were prepared in high yield (>95%). Confocal microscopy images of NCI-H460 cells incubated with GIVA-ECG-ROX revealed strong fluorescence activity in the cytoplasm. In gamma camera imaging, the tumor-to-normal muscle uptake ratios of Tc-99m GIVA-ECG-ROX increased with time (3.8 ± 0.4, 4.5 ± 0.8, and 7.2 ± 1.5 at 1, 2, and 3 h, respectively). Confocal microscopy with immunohistochemistry staining detected significant Tc-99m GIVA-ECG-ROX fluorescence within NCI-H460 tumor tissue. Fluorescent images of mice with peritoneal carcinomatosis indicated the detection of several tumor nodules on the peritoneal surface and in the peritoneal cavity. Under real-time optical imaging, the removal of visible nodules was successfully performed.

-Conclusion
Through in vitro, in vivo, and ex vivo studies, we have demonstrated the specific binding affinity of Tc-99m GIVA-ECG-ROX for PI3K-positive cells and tumors. Notably, following a single administration of Tc-99m GIVA-ECG-ROX, both gamma and fluorescent images could be acquired. These findings strongly indicate the potential of Tc-99m GIVA-ECG-ROX as a promising agent for multimodality imaging targeting PI3K of thyroid malignancy.