Bioluminescent Human Thyrospheres Allow Noninvasive Detection of Anaplastic Thyroid Cancer Growth and Metastases In Vivo

Introduction

Anaplastic thyroid cancer (ATC) is a rare but highly lethal form of thyroid cancer with a mean survival time of less than 6 months from the time of diagnosis. The major challenges of ATC are represented by a lack of diagnostic tools for early detection and a poor response to all current treatment modalities (1,2). ATC is an undifferentiated thyroid cancer that has traditionally been thought to dedifferentiate from well-differentiated thyroid cancers, including papillary and follicular thyroid cancer (3,4). However, emerging evidence indicates that ATC contains a small subset of cancer stem-like cells that have the ability to grow as nonadherent thyrospheres and sustain self-renewal in culture (3,5 –11). We recently evaluated four bona fide human ATC cell lines (THJ-11T, THJ-16T, THJ-21T, and THJ-29T) and showed that approximately 3–9% percent of cells from these cell lines formed thyrospheres when seeded in stem-cell–culture conditions on ultra-low attachment plates. These thyrosphere cells are tumorigenic and, in an orthotopic mouse model of ATC, they metastasize more aggressively than cells derived from the parental monolayer (5). To further our analysis, we describe here the use of bioluminescent human thyrospheres to establish a clinically relevant mouse model of ATC that allows noninvasive and sensitive monitoring of tumor progression and drug response over time.

In recent years, xenografting of firefly luciferase-labeled cancer cells followed by bioluminescent imaging has offered unique optical imaging opportunities to monitor tumor metastases in living animals. Bioluminescence provides a noninvasive and sensitive way to follow cell trafficking and tumor growth in vivo and it has enormous potential when used as a tool to accurately evaluate the outcome of a given pharmacologic intervention. Here, we engineered a luciferase gene into THJ-11T and THJ-16T cell lines to generate stable clones. These clones were used to generate luciferase-expressing thyrosphere cells, which were subsequently orthotopically implanted into the thyroid of NOD/SCIDIl2rg-/- mice to initiate tumors. Tumor progression and metastasis were evaluated by live bioluminescent imaging weekly as well as histologic analysis postmortem.

Materials and Methods

Results

Stable luciferase expression by THJ-11T and THJ-16T cell lines

THJ-11T and THJ-6T cells were transfected with a pSIN-luc vector encoding a firefly luciferase gene (13) and several stable clones from each cell line were selected for further analysis. Three bioluminescent clones from each cell line were chosen for further analyses. The luciferase activities of serial dilutions of cell cultures of each clone were analyzed and compared in vitro. The level of luciferase expression was proportional to the number of cells seeded (Fig. 1).

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FIG. 1.

In vitro bioluminescence of THJ-11T and THJ-16T cells. Stable clones of THJ-11T and THJ-16T cells expressing luciferase were serially diluted in duplicate wells from 50,000 to 1000 cells per well. Luciferin substrate was added to each well 10 minutes before imaging and the plate was imaged to obtain photons/s per cell. Wells with media but not cells, or cells alone without luciferin, were included as controls (A). Note the level of luciferase expression was proportional to the number of cells seeded (B).

Tumor development and metastasis from luciferase-expressing THJ-11T and THJ-16T cells

We next attempted to detect tumor growth and metastasis in vivo of labeled THJ-11T and THJ-16T cells after implantation into groups of NOD/SCIDIl2rg-/- mice (5×10⁵ cells per mouse, n=4 per cell line). This cell number has been shown in our previous studies to be sufficient to cause tumor development in these mice (5). Using an IVIS Spectrum imaging system, we were able to detect bioluminescent signals at the implantation sites in all the mice (Fig. 2). The tumors grew rapidly and developed metastatic lesions that could be detected with bioluminescent imaging as early as 3 days after implantation. They remained detectable until around 33 days, when most of the mice displaying signs of severe cachexia and were sacrificed after losing more than 20% of their body weight. Histologic analysis of the tumors identified many clinical features of ATC—a high-grade malignant neoplasm characterized by a high mitotic index, nuclear atypia, cellular pleomorphism, and necrosis. Mice also developed extrathyroidal invasions to the trachea, smooth muscles, and the esophagus (Fig. 2).

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FIG. 2.

In vivo bioluminescence of THJ-11T and THJ-16T cells in immunodeficient mice. (A) THJ-11T– and THJ-16T-luciferase–expressing cells (500,000 cells per mouse) were injected into the right thyroid of NOD/SCIDIl2rg^-/- mice, and tumor growth and metastasis were monitored over time in vivo. Tumor take rate was 100% (4/4 mice per cell line). (B) Bioluminescent images were taken using an IVIS Spectrum. All mice were reimaged using the same setting of IVIS Spectrum and bioluminescent signals were quantified as total flux photons/s (p/s) using Living Image software and plotted against the days since implantation. (C) Histologic analysis of the tumor tissue confirmed invasion to the trachea, smooth muscles, and the esophagus. Tumors displayed with a mixed morphology of spindle cells and pleomorphic giant cells. Scale bar, 100 μm.

Tumors detected in vivo after intrathyroidal injection of THJ-11T and THJ-16T thyrospheres in mice

After confirming the ability to noninvasively detect bioluminescent THJ-11T and THJ-16T cells in vivo, we attempted to use bioluminescent imaging to detect and track a small number of thyrosphere cells to investigate the hypothesis that ATC contains cancer stem-like cells. We previously demonstrated that 10,000 thyrosphere cells (a cell number 50-fold less than that required when using parental monolayer THJ-11T cells) injected into the thyroid can cause tumor development in mice (5). Here we explored the effect of implanting varying numbers of luciferase-expressing THJ-11T– and THJ-16T–derived thyrosphere cells (100, 1000, and 10,000 cells per mouse, n=4 per cell line) into the thyroids of NOD/SCIDIl2rg-/- mice. Seven days after implantation, all mice developed tumors—a 100% success rate for tumor initiation regardless of the number of cells implanted (Fig. 3). More importantly, we were able to detect tumors arising from as few as 100 cells after only 7 to 14 days. The total flux from the implantation sites was directly proportional to the number of cells implanted (Fig. 3). Histologic analysis of the orthotopic tumors arising from thyrospheres revealed tumors around the trachea and tracheal invasion into smooth muscle and esophagus, and hematoxylin and eosin staining confirmed these tumors are high-grade malignant neoplasms that share characteristics of ATC (Fig. 3). Together, these data clearly show that tumor growth and metastasis in an animal can be monitored using noninvasive bioluminescent imaging. Furthermore, it validates the cancer stem-like cell model of ATC, because even as few as 100 thyrosphere cells are sufficient to develop tumors in mice.

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FIG. 3.

Tumors detected in vivo following intrathyroidal injection of THJ-11T and THJ-16T thyrospheres in mice. (A) THJ-11T– and THJ-16T–luciferase expressing thyrosphere cells were injected into the right thyroid of NOD/SCIDIl2rg-/- mice, and tumor growth and metastasis were monitored over time in vivo. Tumor take rate was 100% (4/4 mice per group). Bioluminescent images were taken weekly. (B) The noninvasive light imaging detected the tumors arising from as few as 100-cell implantation at day 7–14, and the total flux from the implantation sites was directly proportional to the number of cells implanted. (C) Histologic analysis of the tumor tissue confirmed invasion to adjacent tissues and tumors displayed a high-grade malignant neoplasm characterized by necrosis and marked pleomorphism (arrows). Scale bar, 100 μm.

Discussion

Several mouse tumor models of ATC have been established, including subcutaneous and orthotopic xenografts of tumor cells implanted into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening. However, this method is suitable only for palpable tumors growing under the skin of the animals and does not well reflect the metastasis seen in human disease. Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease by implanting tumor cells directly into their equivalent anatomical origin in a host animal. (14,16). However, traditional orthotopic models are impractical for evaluation of drug efficacy since they require large numbers of animals to be sacrificed at each time point. In addition, accurate evaluation and quantitation of tumor burden can be difficult. In contrast, noninvasive whole-body bioluminescent imaging allows us to obtain information in real time without sacrificing the animals and response to treatment can be easily followed longitudinally in a single animal throughout the study. The ability to obtain sequential images of a single animal could yield valuable insight to tumor progression and provide a significant advantage for evaluating therapeutic efficacy in preclinical disease models.

In the present study, we report the first development of mouse models of ATC with bioluminescent human thyrospheres. As few as 100 cells from the ATC thyrospheres were able to form a tumor when orthotopically injected into NOD/SCIDIl2rg-/- mice, whereas at least 5×10⁵ parental cells were needed to generate a tumor in the same model. This was 5000 times higher than that of sphere-forming cells. This model allows the comparison of the effectiveness of a given pharmacologic intervention on early stages of ATC and metastases. It can also serve as a valuable tool for the investigation of biologic processes and the development of novel therapies of ATC in preclinical models.