Thyroid cancer imaging in vivo by targeting the anti-apoptotic molecule galectin-3

Abstract

Background: The prevalence of thyroid nodules increases with age, average 4-7% for the U.S.A. adult population, but it is much higher (19-67%) when sub-clinical nodules are considered. About 90% of these lesions are benign and a reliable approach to their preoperative characterization is necessary. Unfortunately conventional thyroid scintigraphy does not allow the distinction among benign and malignant thyroid proliferations but it provides only functional information (cold or hot nodules). The expression of the anti-apoptotic molecule galectin-3 is restricted to cancer cells and this feature has potential diagnostic and therapeutic implications. We show here the possibility to obtain thyroid cancer imaging in vivo by targeting galectin-3.

Methods: The galectin-3 based thyroid immuno-scintigraphy uses as radiotracer a specific (99m)Tc-radiolabeled mAb. A position-sensitive high-resolution mini-gamma camera was used as imaging capture device. Human galectin-3 positive thyroid cancer xenografts (ARO) and galectin-3 knockout tumors were used as targets in different experiments in vivo. 38 mice with tumor mass of about 1 gm were injected in the tail vein with 100 microCi of (99m)Tc-labeled mAb to galectin-3 (30 microg protein/in 100 microl saline solution). Tumor images were acquired at 1 hr, 3 hrs, 6 hrs, 9 hrs and 24 hrs post injection by using the mini-gamma camera.

Findings: Results from different consecutive experiments show an optimal visualization of thyroid cancer xenografts between 6 and 9 hours from injection of the radiotracer. Galectin-3 negative tumors were not detected at all. At 6 hrs post-injection galectin-3 expressing tumors were correctly visualized, while the whole-body activity had essentially cleared.

Conclusions: These results demonstrate the possibility to distinguish preoperatively benign from malignant thyroid nodules by using a specific galectin-3 radio-immunotargeting. In vivo imaging of thyroid cancer may allow a better selection of patients referred to surgery. The possibility to apply this method for imaging and treatment of other galectin-3 expressing tumors is also discussed.

Figure 1. In vivo detection of galectin-3 positive ARO xenografts by using radio immunoscintigraphy with ^99mTc-labeled mAb to galectin-3.

A) Image acquired with a high-resolution mini gamma camera in a mouse bearing ARO (Gal3+) xenograft after 6 hrs from i.v. injection of 100 µCi of ^99mTc-labelled mAb to Galectin-3. The arrow shows the tumor mass revealed in the left leg. A consistent accumulation of the radio tracer is observed in the liver according to the clearance of exogenous mAb (panel 1A); Morphological and immunohistochemical evaluation of the excised tumor xenograft show a poorly differentiated thyroid cancer with a variable expression of galectin-3, as revealed by a galectin-3 specific mAb and a direct immunoperoxidase staining method (panel 2A); The table shows the kinetic of tumor /normal muscle ratio of the radio tracer at different time points (panel 3A). B) Image acquired with a high-resolution mini gamma camera in a mouse bearing Galectin-3 interfered ARO xenograft (Gal3−) after 6 hrs from i.v. injection of 100 µCi of ^99mTc-labelled mAb to Galectin-3 (panel 1B); Immunohistochemical evaluation of the excised tumor shows a consistent down-regulation of galectin-3 expression (panel 2B); The efficiency of stable galectin-3 RNA interference in down regulating galectin-3 expression is demonstrated in immunoblotting. ARO cells mock-transfected with pSUPER vector were used as control (Ctr); galectin-3 interfered ARO cells were stable transfected with pSUPER-Gal3-551 vector (Gal3i); α-tubulin was used as a loading control. The densitometry analysis of the molecular species visualized in the gel is also shown (panel 3B). Results are expressed as relative densitometry units (rdu), measured normalizing Gal-3 signal with the corresponding α-tubulin band intensity.

Figure 2. Ex-vivo binding of the ^99mTc-labeled mAb to Galectin-3 on papillary thyroid cancer metastasis in a lymph node.

The figure shows the lymph node specimen cut in half longitudinally and imaged by using the mini gamma camera after 1 hr and 2 hrs of incubation with 2 µCi of ^99mTc-labeled mAb to Galectin-3 (0.1 µg protein) in saline solution, in presence (panel B) or not (panel A) of an excess (100 µg) of unlabeled galectin-3 specific mAb. Panel A shows tumour detection by radiotracer after 1 hr and 2 hrs of incubation. Panel B shows a consistent lower uptake of the radiotracer in presence of an excess of unlabelled anti-gal3- mAb, due to the specific displacement effect.