Characterization of human follicular thyroid cancer cell lines in preclinical mouse models

Abstract

Follicular thyroid cancer (FTC) is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in in vivo preclinical models before using them to test new, targeted therapies. This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines (WRO, FTC-238, and TT1609-CO2) with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing.

Keywords: follicular thyroid cancer; metastasis; mouse model; orthotopic; thyroid.

Figure 1.

Morphologies and gene expression analysis of human FTC cell lines. (A) Phase-contrast microscopy images of WRO, FTC-238, and TT2609-CO2 cell lines. (B) Quantitative real-time PCR analysis of PAX8, TTF1, TSHR, TG, TPO, and NIS. Y-axis, relative mRNA expression level compared with normal human thyroid. Human GAPDH was used as the housekeeping gene during the amplification for the normalization of the above gene expression data. The results are expressed as mean ± s.e.m. of two independent experiments with three parallels.

Figure 2.

In vitro bioluminescence of WRO, FTC-238, and TT2609-CO2 cell lines. Stable clones of WRO, FTC-238, and TT2609-CO2 cells expressing luciferase were serially diluted. Luciferin substrate was added to each well 10 min before imaging and the plate was imaged to obtain total flux (p/s) per cell line using IVIS Spectrum. The wells with media but not cells, or cells alone without luciferin, were included as controls. Note that the level of luciferase expression was proportional to the number of cells seeded. The results are expressed as mean ± s.e.m. of two independent experiments with three parallels.

Figure 3.

WRO-derived tumors grew faster than FTC-238 and TT2609-CO2-induced tumors in a murine orthotopic model. Luciferase-tagged WRO, FTC-238, or TT2609-CO2 cells were injected into the thyroids of NSG mice, and tumor initiation and growth were monitored via bioluminescent live imaging weekly using IVIS Spectrum. Bioluminescent tumor signals were quantified as total flux (p/s) using Living image software and plotted against the days since tumor cell injection. The results are expressed as mean ± s.e.m. (n = 2 per group).

Figure 4.

Time course of body weight and in vivo bioluminescence. Upper panel shows body weights of mice injected with WRO, FTC-238, or TT2609-CO2 cell lines. Lower panel shows primary thyroid tumors resulting from injection of WRO, FTC-238, or TT2609-CO2 cells in the orthotopic models as visualized by bioluminescent IVIS imaging. All mice (n = 2 per group) were reimaged using the same setting of the IVIS throughout the study.

Figure 5.

Representative histology images of thyroid tumors and lung metastases in a murine orthotopic model. H&E stained sections of all mice orthotopically injected with WRO, FTC-238, or TT2609-CO2 cell lines show the primary tumor with local invasion to trachea and esophagus (A,D,F). The middle panel shows higher magnification (200x) of tumors from each cell line (B,E,G). Note that mice injected with WRO also developed a lung micrometastasis whereas mice injected with FTC-238 or TT2609-CO2 cells did not ©. Scale bar, 100 mm.

Figure 6.

WRO cells develop lung metastases in the tail vein injection model. Lung metastases were experimentally induced by tail vein injection of 5 × 10⁵ cells. (A) Representative bioluminescent images of mice (n = 2 per group) injected with WRO, FTC-238, or TT2609-CO2 cells taken at day 35. (B) H&E stained sections revealed tumor cells metastasized to the alveoli of the lung in mice injected with WRO cell line. Left panel, 50x magnification; right panel, 200x magnification. Scale bar, 100 mm.