High-Frequency Ultrasound-Guided Injection for the Generation of a Novel Orthotopic Mouse Model of Human Thyroid Carcinoma

Background: Thyroid carcinoma is the most common endocrine malignancy and has an increasing incidence. High-frequency ultrasound (HFUS) has a spatial resolution of 30 lm, which is a property that has been exploited for thyroid visualization and analysis in mice. The aim of this study was to generate a novel orthotopic mouse model of human follicular thyroid carcinoma (FTC) using an HFUS-guided injection system. Methods: Twenty Balb/C nude mice were injected in the right lobe of the thyroid with 2·106 FTC-133 cells using the microinjection HFUS-guided system, and 20 mice, used as a control, underwent surgical orthotopic im- plantation of 2 ·106 FTC-133 cells in the right lobe of the thyroid. All mice underwent HFUS imaging two weeks after cell injection; HFUS examinations and tumor volume (TV) measurements were repeated weekly. Micro– computed tomography was performed at different time points to determine whether lung metastasis had occurred. TVs were compared between the two models (surgical vs. HFUS-guided) using theMann–WhitneyU-test, and the Mantel–Cox log-rank test was applied to evaluate the death hazard. Hematoxylin and eosin analysis of formalin- fixed, paraffin-embedded mouse tissue was performed to validate the in vivo imaging results. Results: Of the HFUS-guided injected mice, 9/18 survived up to 40 days after the injection of tumor cells. Mice injected surgically had 100% mortality at day 29. Of 38 mice, 29 (14/18 HFUS, 15/20 surgical) showed metastasis in the salivary glands and lymph nodes, and 13 (10/18 HFUS, 3/20 surgical) also showed metastasis in the lungs, which was confirmed by histological analysis. In the surgical group, there was an evident, frequent (12/20 mice) involvement of the contralateral lobe of the thyroid, whereas this feature was only detected in 1/18 mice in the HFUS group. Statistical analysis showed the same pattern of growth in the two groups, and a significant hazard in the mice in the surgical group ( p= 0.03). Conclusions: This study demonstrated the technical feasibility of an HFUS-guided orthotopic mouse model of FTC. The HFUS-guided orthotopic model is easily reproducible and allows prolonged monitoring of the disease because the animals showed an increased survival rate. Introduction