Preclinical Models of Follicular Cell-Derived Thyroid Cancer: An Overview from Cancer Cell Lines to Mouse Models
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Abstract
The overall prognosis of thyroid cancer is excellent, but some patients have grossly invasive disease and distant metastases with limited responses to systemic therapies. Thus, relevant preclinical models are needed to investigate thyroid cancer biology and novel treatments. Different preclinical models have recently emerged with advances in thyroid cancer genetics, mouse modeling and new cell lines. Choosing the appropriate model according to the research question is crucial to studying thyroid cancer. This review will discuss the current preclinical models frequently used in thyroid cancer research, from cell lines to mouse models, and future perspectives on patient-derived and humanized preclinical models in this field.
INTRODUCTION
Thyroid cancer is the most common endocrine malignancy and generally has an excellent prognosis. The overall 10-year survival rate is over 95%, but 5% to 10% of patients have distant metastatic disease, and one-third of these patients have radioactive iodine resistance with progressive disease [1,2]. Anaplastic thyroid cancer (ATC) is the most dedifferentiated and lethal of all cancers. Median survival of patients with ATC was reported as 5 to 6 months, even with multimodal treatment [3]. Over the past few years, targeted therapies have shown significant clinical benefit for patients with differentiated thyroid cancer (DTC) and ATC [4-7]. However, these therapies are not curative, and most patients eventually progress, underlying the critical need for more effective therapies [8]. Recent studies using immunotherapies also showed limited efficacy [9-12]. Preclinical experimental models are crucial for investigating thyroid cancer pathogenesis and developing novel effective therapies.
Recent progress in genetics and molecular biology has led to the development of many preclinical models with genetic characterization of these models [2,13,14]. Choosing the suitable model to ask specific questions is critical in understanding thyroid cancer pathogenesis and treatment. This review focuses on the preclinical models of follicular cell-derived thyroid cancer. We will provide the overview of available thyroid cancer cell lines and mouse models with a summary of their characteristics, advantages, and disadvantages.
CELL LINES
Cancer cell lines are the most widely used tool for in vitro and in vivo cancer research. Many thyroid cancer cell lines from different origins with various genetic characteristics are available for research, and the genomic landscape of the cell lines is well established (Table 1) [15]. It is critical to get cell lines form a trusted source that carefully validates the cell lines on a regular basis [16]. The University of Colorado Cancer Center has a repository of many of the authenticated thyroid cancer cell lines (https://medschool.cuanschutz.edu/colorado-cancer-center/research/shared-resources/cell-technologies/cell-culture-services). Thyroid cancer cells have driver mutations similar to those in primary human tumors: BRAF V600E is the most common driver mutation, and mutations in RAS genes are also frequently observed. However, thyroid cancer cell lines are profoundly dedifferentiated and have gene expression profiles similar to ATCs even though many originated from differentiated cancers. Furthermore, telomerase reverse transcriptase (TERT) promoter and tumor protein p53 (TP53) mutations appear to have been acquired or enriched under cell culture conditions [15].
Commercially Available Thyroid Cancer Cell Lines according to the Origin of Tumor and Genetic Characteristics
Cancer cell lines have a significant limitation as they acquire genetic changes needed for immortalization and are not always representative of the original tumor. There are also issues of cross-contamination with other cell lines and infection with mycoplasma that can affect cell metabolism and other properties. Furthermore, the effect of the tumor microenvironment can’t be assessed in these cancer cell lines [15,17]. However, cell lines are useful for large drug screens, and understanding the effects of cell signaling on proliferation, apoptosis, migration/invasion and anchorage-independent growth [15,17]. Cell lines are also generally easy to grow and provide a nearly limitless research tool.
To overcome some of the problems of cancer cell lines, researchers have established organoid models or cell lines from patient-derived xenografts (PDXs) that show gene expression profiles similar to the original tumor [18-20]. Papillary thyroid carcinoma (PTC) organoids also preserve most of the microenvironment of the originating tumors and better approximate patient drug responses [20].
MOUSE MODELS
Preclinical animal models are critical in cancer research, and the mouse model is considered most useful due to their short lifespan, small body size, and genetic similarities to humans [21]. In the past, thyroid cancer studies were mainly performed in vitro with thyroid cancer cell lines, then confirmed in vivo using immunosuppressed xenograft models. The traditional technique of the xenograft model has been subcutaneous human tumor implants on the flank of an immunocompromised mouse. More recently, investigators have used orthotopic implants of human thyroid cancer cells into the thyroid bed as well as intracardiac injections in immunocompromised mouse [22,23]. Currently, genetic manipulation using the Cre-loxP system developed various genetically engineered mouse models (GEMMs) for thyroid cancer, and the mouse models continue to evolve [2,24]. The summary of each mouse model’s characteristics is shown in Table 2 and the advantages and challenges of different types of mouse models are summarized in Table 3.
Characteristics of Mouse Models of Cancer
The Advantages and Challenges of Different Types of Mouse Models for Thyroid Cancer
Xenograft models
The xenograft model is established by implanting human cancer cell lines or tissues into immunodeficient mice, which can preserve genetic alterations of human cancers [25,26]. According to the location where those were implanted, xenograft models are further classified into ectopic (subcutaneous), orthotopic (thyroid bed), and metastatic (tail vein or intracardiac) [27,28]. The most commonly used immunocompromised mice are athymic nude, severely combined immune-deficiency (SCID) and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice [19,22,27].
The flank xenograft model is relatively easy to generate and monitor tumor growth and treatment response. However, this model lacks the local tumor microenvironment and distant metastasis [26]. The orthotopic xenograft model is more physiologic and tumorigenic than the flank models and has distant metastatic potential [25,27]. Nucera et al. [22] generated the 8505C orthotopic tumor which showed palpable neck tumors at 20 days post-transplantation and miliary multifocal lung micrometastasis. Morrison et al. [28] generated orthotopic tumors using various thyroid cancer cell lines and reported different take rates or tumor volumes. 8505C, T238, and K1 cell lines showed 100% take rate, but HTH7, C643, SW1736, MDA-T41, and TPC-1 cells failed to form orthotopic tumors. The orthotopic xenograft model also has an advantage in stimulating thyroid stromal cells, and the local innate immune system [25]. Some limitations of this model include rapid growth, which is different than many relatively slow growing advanced human cancers, the lack of an adaptive immune system and the deposition of cell not only in the thyroid, but around the thyroid, making assessment of extrathyroidal invasion difficult.
Patient-derived xenograft models
PDX models directly use patient-derived tissues, recreate human cancer gene expression, tumor heterogeneity, and microenvironment, and are expected to inform drug response better than standard xenograft models [19]. These models are successfully established in many cancers but are still limited in thyroid cancer research. Maniakas et al. [19] generated PDX model using six ATC tumors: the tumor implanted in the flank of immunodeficient athymic mice and expanded for at least four generations. Mutation, histopathological characterization of PDX models showed good fidelity with the original tumor. The take rate was about 30% [19]. This group of researchers reported the result of high-throughput screening multiple drugs using these models and matching cell lines and suggested the feasibility of using this systematic approach for preclinical in vivo drug testing or personalized therapeutics development [29].
Syngeneic models
The syngeneic model is established by implanting mouse cancer cells or tissues into immunocompetent mice of the same genetic background. Like the xenograft model, the syngeneic model can be ectopic (subcutaneous), orthotopic (thyroid bed), or metastatic (tail vein or intracardiac) [21]. Vanden Borre et al. [30] reported the development of syngeneic ATC orthotopic mouse model using cell lines derived from tumors arising in GEMM with thyroid-specific expression of mutant BRAF and deletion of either TP53 or phosphatase and tensin homolog (PTEN), which were implanted into immunocompetent syngeneic B6129SF1/J mice. Caperton et al. [31] reported the development of syngeneic follicular thyroid cancer (FTC) ectopic mouse model using tumor cells from GEMM with mutant HRas and PTEN inactivation. These models showed very rapid tumor formation with significant immune cell infiltration in the tumor [30-32]. The syngeneic model has a significant advantage over the xenograft model in that researchers can study tumor growth with an intact immune system and investigate the immunemodulatory effect of therapies. The short latency of tumor development also makes this an attractive model [21,30]. The major drawback of this model is that the immunoediting process, so critical in human tumor development, may not proceed due to very rapid tumor formation, which introduces a model bias in the response of immunotherapeutic agents [33]. Because of very rapid tumor growth in most syngeneic mouse models, distant metastasis cannot be easily studied [30].
Genetically engineered models
The GEMM is established by genetic engineering tools and the Cre-loxP system is the most widely used [34]. The expression of oncogenes and/or loss of tumor suppressor genes can be spatially edited by controlling the expression of Cre recombinase using cell-specific regulatory elements such as thyroglobulin (Tg) or thyroid peroxidase (TPO), as well as temporally controlled by further engineering of Cre and exogenous inducers like tamoxifen (Table 4) [35].
Summary of Genetically Engineered Mouse Models for Follicular Cell-Derived Thyroid Cancer
As BRAF V600E mutations account for the majority in PTC and ATC tumors, many GEMMs contain BRAF V600E [2]. The first BRAF GEMM used the Tg promoter and constitutive BRAF V600E cDNA overexpression resulting PTC with high penetrance. However, the BRAF transgene led to dedifferentiation, which exhibited a loss of Tg expression, causing a negative feedback loop on the Tg-BRAF transgene [36]. To overcome this, a subsequent model used tamoxifen-inducible Cre (CreERT2) under the control of the Tg promoter and Cre-activating BRAF V600E (BrafCA) allele. PTCs were seen 6 months after tamoxifen injection and progressed to advanced DTC at 12 months. However, distant metastases were not detected [37]. Chakravarty et al. [38] also reported that conditional activation of BRAF V600E with doxycycline in the thyroid under the control of the Tg promoter resulted in mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) activation and PTC formation after 1 week of drug exposure. The treatment with a MEK inhibitor in this model restored tumor radioiodine uptake [38], leading to many human studies exploring BRAF and MEK inhibition to improve radioiodine uptake in humans with DTC [39,40]. All these BRAF tumor models showed decreased thyroid function and elevated thyroid stimulating hormone (TSH) due to dedifferentiation [36-38,41]. Many investigators treat these mouse models with levothyroxine to mitigate the effect of high TSH on the model and better approximate the human condition. McFadden et al. [42] reported that BRAF V600E initiates PTC in the adult mouse thyroid and additional TP53 loss enables the progression to ATC. PTC tumors developed in mice with TPO-CreERT2/BrafCA/+ at 12 weeks postinduction. Crossing this strain with the mice with homozygous deletion of p53 (Trp53f/f) further accelerated to overt ATC. This model is referred to as TBP. An explosive tumor growth was seen approximately 100 days after tamoxifen treatment. They also showed that the selective BRAF inhibitor was not effective in controlling these ATC tumors and the combination of MEK/BRAF inhibitor was more effective [42]. Additional activation of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3KCA) (Tg-CreERT2/BrafCA/PIK3CAH1047R) or knock-out of PTEN (Tg-CreERT2/BrafCA/PTENf/f) also resulted in the development of ATCs [43].
RET/PTC or neurotrophic tyrosine receptor kinase (NTRK) rearrangement are also drivers of PTC [44,45]. The RET/PTC1 [46,47] and RET-PTC3 [48] transgenic mouse models using Tg promoter were reported, and both resulted in the development of PTC. However, these RET/PTC rearrangement in mice are germline, and PTC development and tumor progression may be more advanced in these mouse models compared with sporadic genetic alterations in humans. Buckwalter et al. [49] reported that mutation of major tyrosine signaling sites in RET/PTC1 transgenic mice didn’t block the development of the tumor. This suggests that other signaling pathways, or the microenvironment may play a major role in tumor formation [49]. The fusion of translocated promoter region (TPR) with NTKR1 (TRK-T1) developed follicular hyperplasia or PTC in about 50% of the mice [50].
RAS activation is a well-known driver in FTC. An early mouse model containing a mutant K-RasG12V gene under the control of rat Tg promoter showed no thyroid cancer development, and only one of these mice developed FTC following treatment with goitrogens (aminotriazol and sodium perchlorate in the drinking water) for 6 months [51]. This result suggests that RAS mutation alone or this degree of RAS signaling is insufficient to induce FTC and other alterations are required. One study using transgenic mice harboring the human N-RasQ61K under the control of the bovine Tg promoter reported that approximately 40% of the mice developed invasive FTC, in some with a mixed papillary morphology and about 25% showed de-differentiation with distant metastases [52]. These data suggest that secondary mutations may occur, or multiple copies of the transgene could be incorporated and lead to the abnormally high transcript expression [24]. These older transgenic mouse models of FTC have a limitation in that the cancer phenotype resulting from overexpression of a mutated RAS gene is different from the endogenous mutant RAS expressed at the physiological level. Miller et al. [53] crossed a mouse strain with TPO-Cre and an oncogenic K-RasG12D which is conditionally expressed through Cre-mediated deletion of a floxed STOP cassette preventing K-RasG12D expression (lox-stop-lox [LSL]-K-Ras), with a mouse strain with PTEN loss (PTENf/f). All of the double-mutant mice (TPO-Cre/LSL-K-RasG12D/PTENf/f) rapidly developed FTC and none survived over age 4 months [53]. Thyroid-specific double protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A)-PTEN knock-out mice also developed FTC [54]. Serum TSH levels were suppressed and thyroxine was elevated in these double-mutant mice, indicating development of thyrotoxicosis from functional FTC [53,54]. Thyroid-specific expression of H-RasG12V and PTEN inactivation (TPO-Cre/H-RasG12V/PTENf/f) also leads to the development of high grade FTCs or poorly differentiated thyroid cancer (PDTC) showing gross extrathyroidal extension and/or lymphovascular invasion, and lung metastasis [55].
A dominant negative mutation (PV) into the thyroid hormone nuclear receptor β (TRβ) generates another FTC mouse model. TRβPV/PV mice reproduce human thyroid hormone resistance syndrome. T3 is unbale to bind mutant TRβ, and follicular cells become hyperplastic with elevated TSH levels. Invasive FTC was observed at 4 to 5 months of age and distant metastases to lung or heart was seen over 5 months of age. TRβPV/− mice from crossing TRβPV/PV mice with TRβ−/− mice also showed spontaneous FTC with lung metastasis [56]. The phosphoinositide 3-kinase (PI3K)-AKT signaling was activated in this model and PI3K inhibitor treatment inhibited FTC progression in these mice [57].
The GEMM model best interrogates the tumor microenvironment and the interaction between the tumor and immune system because the tumor is developed more slowly over time, not grafted [25,26,33]. Furthermore, this model helps to dissect the effect of each genetic alteration on tumor initiation and progression [2]. This model has limitations including long latency to tumor formation, complex breeding schemes, and high costs [25].
CONCLUSION AND FUTURE PERSPECTIVES
These different preclinical models have greatly improved our understanding of thyroid carcinogenesis and the development of new therapeutic strategies. Thyroid cancer cell lines and mouse models have different strengths and challenges, and any single model can’t accurately reproduce all features found in human cancer. Understanding the characteristics and challenges of each model and applying the best model to specific research questions is important.
The humanized mouse model is one of the newer techniques in cancer research [58] and could be an important future preclinical model in thyroid cancer research. This model is established by transplanting human-derived peripheral polymorphonuclear cells or hematopoietic stem cells into severe combined immunodeficient mice to promote the development of the functional human immune system in mice. This model can reproduce the human tumor microenvironment, enabling a better to study immunotherapy. However, this model still faces many challenges, including frequent engraftment failure, MHC incompatibility between immune cells and tumors, the residual murine innate immunocytes, and the lack of specific cytokines [58]. These future models, including more advanced GEMM, PDX, and organoid models, will help bench-to-bedside studies in thyroid cancer research.
Notes
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was reported.