Galectin-3 targeting in thyroid orthotopic tumors opens new ways to characterize thyroid cancer

Abstract

Preoperative characterization of thyroid nodules is challenging since thyroid scintigraphy fails to distinguish between benign and malignant lesions. Galectin-3 is widely expressed in the majority of well-differentiated thyroid carcinomas and in a large fraction of undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. Herein, we aimed to validate galectin-3 targeting in thyroid orthotopic tumors otherwise not visible with conventional imaging. Methods: Papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cells lines were characterized via WB and PCR, for galectin-3 and sodium iodide symporter (NIS) expression. A 89Zr-labeled F(ab’)2 anti-gal3 was generated and characterized for binding versus iodine-125 on 2D and 3D cell cultures. Thyroid orthotopic murine models were established inoculating carcinoma cells into the left thyroid lobe of athymic nude mice, and the tumor growth was monitored via ultrasound and fluorescence molecular tomography (FMT). Head-to-head PET/CT comparison of iodine-124 versus 89Zr-DFO-F(ab’)2 anti-gal3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. Results: The thyroid carcinoma cells investigated were invariably Galectin-3 positive, while presenting low/lost NIS expression. 89Zr-DFO-F(ab’)2 anti-Gal3 tracer showed high stability, high affinity to Gal3 (Kd ~3.9 nM), retained immunoreactivity (>75%) tested on 2D cell cultures and on tumor spheroids, while iodine-125 internalization in FRO82-1, BcPAP and CAL62 was related to NIS expression, both in 2D and tumor spheroids. In vivo PET/CT imaging showed excellent image contrast for the 89Zr-DFO-F(ab’)2 anti-Gal3 with signal associated to the orthotopic-implanted tumors while no signal was present in the tumor-free thyroid lobe. Conversely, PET imaging of iodine-124 showed a low to no accumulation in tumor bearing lobe based on low/lost NIS expression, a condition simulating the presence of radioiodine non-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe only. Imaging data were confirmed by tracer biodistribution studies and immunohistochemistry. Conclusion: A specific and selective visualization of thyroid tumor by targeting galectin-3 was demonstrated in absence of radioiodine uptake. Translation of this method into clinical setting promises to improve the management of patients, by avoiding the use of unspecific imaging methodologies and reducing unnecessary thyroid surgery.