| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Human Cancer Biology |
Departments of 1 Head and Neck Surgery and 2 Biostatistics, University of Texas M.D. Anderson Cancer Center, Houston, Texas
Requests for reprints: Jeffrey N. Myers, Department of Head and Neck Surgery, University of Texas M.D. Anderson Cancer Center, Unit 441, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713-792-6920; Fax: 713-794-4662; E-mail: jmyers{at}mdanderson.org.
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experimental Design: Various thyroid carcinoma cell lines were injected into the thyroid gland of athymic nude mice to determine whether such injection was technically feasible. ATC cells were then injected into the thyroid gland or the subcutis of nude mice at various concentrations, and the mice were then followed for tumor development. The tumors were examined histopathologically for local invasion or regional or distant metastasis.
Results: Injection of tumor cells into the thyroid glands of nude mice was technically feasible and resulted in the formation of thyroid tumors. The ATC cell line DRO showed significantly higher tumorigenicity in the thyroid gland than in the subcutis. In contrast, oral squamous cell carcinoma cell line TU167 shows no significantly higher tumorigenicity in the thyroid gland than in the subcutis. ATC tumors established in the thyroid gland also produced symptomatic compression of the esophagus and the trachea. Local invasion of the larynx and trachea was as well as high rates of pulmonary metastasis were also observed. Immunohistochemical staining showed higher microvessel density as well as higher expression of vascular endothelial growth factor and interleukin-8 in the orthotopic thyroid tumors than in ectopic tumors.
Conclusion: An orthotopic model of ATC in athymic nude mice was developed that closely recapitulates the clinical findings of human ATC. This model should facilitate the understanding of the pathogenesis of ATC and aid in the development of novel therapies against ATC.
Key Words: preclinical study • Interleukin-8 • Vascular endothelial growth factor
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
8 cm (3). Furthermore, 40% and 50% of patients already have regional and distant metastasis, respectively, at the time of diagnosis (4). Inherent in the extremely poor prognosis of patients with ATC is the absence of any effective curative modality against this disease. Although surgical resection, chemotherapy, or radiation therapy may be employed in the treatment of ATC, they are most often used with palliative rather than curative intent. The development of effective curative treatments for patients with ATC has been hampered by its relative rarity. Another major hurdle in this endeavor has been the lack of an appropriate animal model in which potential therapeutic strategies against ATC may be evaluated. Most current animal models consist of establishing s.c. tumors in nude mice. However, it has been shown that the microenvironment of the subcutis is radically different from that of the organs where tumors originate (5–7). This difference manifests in the different biological behavior of the tumors in orthotopic versus ectopic locations (8–11). Indeed, tumors in orthotopic locations have been shown to have significant differences in drug response compared with tumor established s.c. (9, 10). Furthermore, s.c. tumors in mice fail to display metastatic patterns reflective of the original human tumors (7). In contrast, orthotopic models accurately replicate the metastatic process and have proven to be valuable for selecting populations of tumor cells with increased metastatic potential via serial passages of lymph node metastases (12, 13).
It is now well established that orthotopic tumor cell implantation in nude mice is the method of choice for modeling human cancers because this model allows the evaluation of treatment strategies in a biological setting that closely mimic the disease process in humans. To facilitate the investigation of effective therapeutic strategies for ATC and to better understand its mechanisms of tumor progression, we have developed an orthotopic model of ATC in nude, athymic mice. This model closely replicates the clinical behavior of ATC in humans, including rapid tumor growth, tracheal and esophageal compression, laryngeal and tracheal invasion, cachexia, and morbidity due to obstruction of upper aerodigestive tract, and high incidence of local and distant metastasis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Animal Care and Injection. Eight- to 12-week-old male athymic nude mice were purchased from the National Cancer Institute (Bethesda, MD) and housed in a specific pathogen-free animal facility. The animals were fed irradiated mouse chow and autoclaved reverse osmosis–treated water. All of the animal procedures were done in accordance with a protocol approved by the Institutional Animal Care and Use Committee.
Before injection of tumor cell suspension, the mice were first anesthetized with i.p. injection of sodium pentobarbital (50 mg/kg). Cells (5 x 105) from ARO, DRO, and C643 cell lines were suspended in RPMI 1640 at a final dilution of 1 x 105 cells/µL, and 5 µL (5 x 105 cells) of that suspension was injected into the thyroid glands of each mouse. Each cell line was injected into five mice. These mice were killed by CO2 inhalation 2 weeks after the injections, and necropsy was done to examine for the presence of tumors.
After confirming that injection of thyroid carcinoma cell lines into the thyroid gland of nude mice was technically feasible and resulted in tumor development, we specifically examined the tumorigenicity of the ATC cell line DRO and squamous cell carcinoma of the oral cavity cell line TU167 in the thyroid gland and the subcutis. DRO and TU167 cells were then injected into the thyroid gland and the subcutis of the right flank at the following concentrations: 5 x 105, 1 x 105, 2.5 x 104, 5 x 103, and 10 x 103 cells. The injection volume was 5 µL (5 x 105 cells) for each mouse. A group of five mice were injected for each dose level. The mice were examined for the presence of tumor every other day by manual palpation and gross visual inspection. The presence of tumor was verified by necropsy and by histologic confirmation. The weights of the mice were measured twice a week. The mice were sacrificed if moribund or for weight loss of >20% of their preinjection body weight. Otherwise, the mice were sacrificed 6 weeks after tumor cells injection by CO2 inhalation. All mice underwent necropsy regardless of whether they were killed at the end of the study or during the study.
Orthotopic Thyroid Injection Technique. The entire injection procedure was done with the aid of a dissecting microscope. After administration of sodium pentobarbital, a midline cervical incision was made. The underlying submandibular glands were retracted laterally and the central compartment of the neck was visualized (Fig. 1A and B). Gentle retraction of the midline strap muscles revealed the thyroid gland adjacent to the trachea and visible underneath a deeper layer of semitransparent strap muscles (Fig. 1C). Direct injection of the right thyroid gland was then done using a 25 µL Hamilton syringe (Hamilton Company, Reno, NV) and 30-gauge hypodermic needle. The injection volume was 5 µL. The submandibular glands were returned to the original location and the skin was closed in a single layer with the use of staples.
|
Necropsy and Tissue Preparation. After the mice were killed, the cervical lymph nodes, lungs, and thyroid gland in continuity with the larynx and trachea were removed and placed in formalin solution overnight. Each specimen was embedded in paraffin and then sectioned. The sections were stained with H&E and evaluated by light microscopy for the presence of regional or distant metastasis.
Immunohistochemical Staining on Tumor Tissues. To determine the intratumoral microvessel density, staining with antibodies against tumor endothelium was done using rat anti-mouse CD31/platelet/endothelial cell adhesion molecule 1 antibody (PharMingen, San Diego, CA). Staining for interleukin-8 (IL-8) was done using rabbit anti IL-8 antibody (Biosource, Camarillo, CA).
For CD31/platelet/endothelial cell adhesion molecule 1 and IL-8 staining, fresh frozen tumors were sectioned (8-10 mm thick), mounted on positively charged Superfrost slides (Fisher Scientific, Houston TX), air-dried for 30 minutes, and fixed in cold acetone for 10 minutes. The slides were washed thrice with PBS (pH 7.5), blocked for 20 minutes at room temperature in PBS supplemented with 1% normal goat serum and 5% normal horse serum (protein-blocking solution), and incubated with primary antibody (diluted 1:800 for CD31/platelet/endothelial cell adhesion molecule 1 and 1:50 for IL-8) for 18 hours at 4°C. Samples were then washed thrice for 3 minutes with PBS and blocked with protein-blocking solution for 10 minutes, and then incubated with goat anti-rat IgG conjugated to horseradish peroxidase (Molecular Probes, Eugene, OR) diluted at 1:400 or goat anti-rabbit horseradish peroxidase (Molecular Probes) diluted at 1:400 for 1 hour at room temperature. The slides were washed again in PBS thrice followed by incubation with 3,3'-diaminobenzidine for 10 minutes. The slides stained for IL-8 were counterstained with Gill's hematoxylin 3 after the degree of staining was quantified as outlined below. The slides stained for CD31/platelet/endothelial cell adhesion molecule 1 were counterstained with Gill's hematoxylin 3 directly after incubation in 3,3'-diaminobenzidine.
To stain for basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), paraffin-embedded sections were first dewaxed in xylene. Excess xylene was removed by washing the slides in ETOH. After treating the tissue with pepsin for 20 minutes at 37°C, the slides were washed thrice with PBS. Endogenous blocking was done with 3% H2O2 followed by protein blocking using 5% horse serum with 1% goat serum (protein blocking solution). Rabbit anti-human VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:500 and rabbit anti-human bFGF antibody (Sigma, St. Louis, MI) diluted 1:1,000 were added to the slides for 18 hours at 4°C. The slides were then washed with PBS thrice, blocked again with protein blocking solution for 1 hour, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:500 dilutions for 1 hour at room temperature. The slides were washed again in PBS thrice followed by incubation with 3,3'-diaminobenzidine for 10 minutes. The degree of staining was then quantified for each slides as outlined below. After quantification of staining, the slides were counterstained with Gill's hematoxylin 3.
Quantification of Basic Fibroblast Growth Factor, Vascular Endothelial Growth Factor, and Interleukin-8 Staining Intensity and Determination of Microvessel Density. For quantification of the immunohistochemical reaction intensity, the absorbance of bFGF-, VEGF-, and IL-8-positive cells from either orthotopically established or ectopically established ATC tumors were measured in random 0.039 mm2 fields at x100 magnification using the Optimas Image Analysis software (Silver Spring, MD). Five tissue sections were stained for each group and four fields were evaluated for each section. The samples were not counterstained, so that the absorbance was attributable solely to the product of immunohistochemical reaction. To quantify the microvessel density, four 0.159 mm2 fields at x100 magnification were captured for each tumor section, and CD31-positive microvessels were quantified according to methods described previously (14, 15) . Five tissue sections were stained for each group and four fields were evaluated for each section.
Microscopy. Stained sections were examined in a Microphot-FX microscope (Nikon, Melville, NY) equipped with a three-chip-charged couple device color video camera (Model DXC990, Sony Co., Tokyo, Japan). The photomontages were prepared using Photoshop software (Adobe Systems, Inc., San Jose, CA).
Statistical Analysis. Because of the relatively small sample size of these experiments and the pattern of observed tumor growth, statistical methods based on standard maximum likelihood based methods were not applicable. As an alternative, penalized logistic regression was used to assess the effects of thyroid versus s.c. inoculation (whereas accounting for the effects of the cell dilution) on tumorigenicity. The regression model included terms for the dilution effect and for the inoculation location (thyroid versus s.c.). Differences in survival times were assessed using the log-rank test. Group differences for continuous measurements were assessed via the nonparametric Kruskal-Wallis test or the nonparametric Wilcoxon rank sum test. Ps for the location effect were evaluated against a 
significance level of 0.05. Statistical analysis for comparing microvessel density, bFGF, VEGF, and IL-8 staining between orthotopic and ectopic ATC tumors was done with Student's t test using SPSS software.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Tumorigenicity of the Squamous Cell Carcinoma of the Oral Cavity Cell Line TU167 when Injected into the Thyroid Gland and Subcutis of Nude Mice. It is possible that the thyroid gland of nude mice is generally more permissive to tumor formation than the subcutis, regardless of the organ of origin of the injected tumor cell line. It is also possible that the local injury introduced during the thyroid injection procedure may create an environment more permissive to tumor formation. To assess this possibility, the squamous cell carcinoma of the oral cavity cell line, TU167, was injected into the thyroid glands and the subcutis of nude mice at 5 x 105, 1 x 105, 2.5 x 104, 5 x 103, and 1 x 103 cells per animal. It has been shown previously that TU167 efficiently forms tumors when injected at the above range of concentrations into its orthotopic site (tongue) in nude mice (16). However, none of the animals inoculated s.c. with TU167 developed tumors, regardless of the concentration of injected cells. More importantly, the tumorigenicity of TU167 was only marginally higher in the thyroid gland where tumors developed in only two of five animals inoculated with 5 x 105 cells per animal. In contrast to the DRO cell line, the difference in tumorigenicity for TU167 in the thyroid gland and the subcutis was not statistically significant (P = 0.18).
Orthotopic Model of Anaplastic Thyroid Carcinoma in Nude Mice Recapitulates the Clinical Features of Human Anaplastic Thyroid Carcinoma. One of the striking features of the ATC is its rapid and invasive growth at the primary site. Animals inoculated with DRO cell line showed rapid tumor growth causing tracheal and esophageal compression (Fig. 3). The mice became moribund and cachetic within 1 to 2 weeks following the injection. At the time of necropsy, compression of the trachea and esophagus were confirmed. The differences in survival duration of the animals were directly related to the concentration of the injected cells (Fig. 4) and were statistically significant. The mean survival durations were 18, 26, and 31 days for animals injected with 5 x 105, 1 x 105, and 2.5 x 104 cells per animal, respectively.
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The site-specific interaction between the tumor cells and the host organ has been shown to influence the phenotype of tumor cells in various aspects. It has been shown previously that sensitivity to chemotherapeutic drugs differs between orthotopically established tumors and the same tumor established ectopically (10, 11). Furthermore, the interactions between the tumor cells and the host organs influence the expression of autocrine and paracrine growth factors as well as their receptors (8, 11). In this study, ATC cell line DRO showed significantly higher tumorigenicity when injected into the thyroid gland, and the orthotopic tumors showed higher microvessel density than ectopically established ATC tumors. This increase in microvessel density was accompanied by a corresponding increase in tumor production of VEGF and IL-8. These cellular and molecular differences between orthotopic and ectopic ATC tumors most likely represent the restoration of tumor-host organ interaction that is lost at the ectopic sites.
The major utility of orthotopic cancer models is in recapitulation of the metastatic potentials and patterns seen in human cancer. This requirement of an orthotopic model in promoting metastasis arises from both anatomic and biological constraints. First, the lymphatic drainage and the vascular supply of the subcutis are vastly different than the orthotopic organ. Therefore, the s.c. xenograft model cannot reproduce the patterns of regional and distant metastasis that are characteristic of a particular human cancer. Second, the biological differences between the orthotopic and ectopic location also contribute to the extremely low metastatic rates of s.c. established tumors. Therefore, highly metastatic cancer models have been established in nude mice for carcinomas of the bladder, stomach, pancreas, lung, and the oral tongue by inducing these tumors orthotopically (12, 13, 16, 18, 19). Likewise, our orthotopic model of ATC produced cervical lymphatic metastasis within the regional draining lymphatic basin. This model also produced high rates of pulmonary metastasis that are reflective of human ATC.
Another advantage of an orthotopic model of cancer is its ability to reproduce the site-specific spectrum of clinical findings and the effects these findings have on survival. As seen in patients with ATC, orthotopically produced ATC in nude mice produced high incidence of tracheal and esophageal compression as well as direct laryngeal/tracheal invasion. As result, the animals became moribund and required sacrifice within 3 to 4 weeks after tumor cell injection. Upon necropsy, obstruction of upper aerodigestive tract was confirmed to be the cause of the morbidity. These features of the orthotopic model will allow the evaluation of antitumor compounds in its ability to prevent the local extension of the disease and also to influence survival.
For any animal model to be useful, it must be technically feasible and relatively easy to perform. Injection of the thyroid glands can be done in 2 to 3 minutes per mouse. The procedure was also well tolerated by the animals with post-procedure mortality of <1%. Also, by performing only unilateral injection of the thyroid gland, any iatrogenic morbidity such as hypothyroidism, hypoparathyroidism, or bilateral vocal cord paralysis were prevented.
Lastly, as with any animal model, the limitations of this orthotopic model must be considered. Although the use of orthotopic site is a vast improvement in disease modeling over the use of ectopic sites (s.c.), the biology and physiology of immunodeficient mice does not correlate directly with that of human. Where possible, findings derived from this model should be additionally substantiated with studies in immunocompetent models and analysis of human tumor specimens.
In conclusion, we have developed an orthotopic model of ATC in nude, athymic mice that reproduces the clinical and pathologic features of ATC in human. This orthotopic model will facilitate the development and evaluation of novel therapeutic compounds for the treatment of this almost uniformly fatal disease. Furthermore, the model should allow further progress in enhancing our understanding into the molecular and cellular mechanisms that underlie the pathogenesis and metastasis of ATC.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9/16/04; revised 11/21/04; accepted 12/ 2/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S.-H. Ahn, Y. Henderson, Y. Kang, C. Chattopadhyay, P. Holton, M. Wang, K. Briggs, and G. L. Clayman An Orthotopic Model of Papillary Thyroid Carcinoma in Athymic Nude Mice Arch Otolaryngol Head Neck Surg, February 1, 2008; 134(2): 190 - 197. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Gomez-Rivera, A. A. Santillan-Gomez, M. N. Younes, S. Kim, D. Fooshee, M. Zhao, S. A. Jasser, and J. N. Myers The Tyrosine Kinase Inhibitor, AZD2171, Inhibits Vascular Endothelial Growth Factor Receptor Signaling and Growth of Anaplastic Thyroid Cancer in an Orthotopic Nude Mouse Model Clin. Cancer Res., August 1, 2007; 13(15): 4519 - 4527. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kim, Y. D. Yazici, G. Calzada, Z.-Y. Wang, M. N. Younes, S. A. Jasser, A. K. El-Naggar, and J. N. Myers Sorafenib inhibits the angiogenesis and growth of orthotopic anaplastic thyroid carcinoma xenografts in nude mice Mol. Cancer Ther., June 1, 2007; 6(6): 1785 - 1792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Nahari, R. Satchi-Fainaro, M. Chen, I. Mitchell, L. B. Task, Z. Liu, J. Kihneman, A. B. Carroll, L. S. Terada, and F. E. Nwariaku Tumor cytotoxicity and endothelial Rac inhibition induced by TNP-470 in anaplastic thyroid cancer Mol. Cancer Ther., April 1, 2007; 6(4): 1329 - 1337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. B. Knostman, S. M. Jhiang, and C. C. Capen Genetic Alterations in Thyroid Cancer: The Role of Mouse Models Vet. Pathol., January 1, 2007; 44(1): 1 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Shu, B. Antalffy, J. M. F. Su, A. Adesina, C.-N. Ou, T. Pietsch, S. M. Blaney, C. C. Lau, and X.-N. Li Valproic Acid Prolongs Survival Time of Severe Combined Immunodeficient Mice Bearing Intracerebellar Orthotopic Medulloblastoma Xenografts Clin. Cancer Res., August 1, 2006; 12(15): 4687 - 4694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wang, G. Chakravarty, S. Kim, Y. D. Yazici, M. N. Younes, S. A. Jasser, A. A. Santillan, C. D. Bucana, A. K. El-Naggar, and J. N. Myers Growth-Inhibitory Effects of Human Anti-Insulin-Like Growth Factor-I Receptor Antibody (A12) in an Orthotopic Nude Mouse Model of Anaplastic Thyroid Carcinoma Clin. Cancer Res., August 1, 2006; 12(15): 4755 - 4765. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Younes, Y. D. Yazici, S. Kim, S. A. Jasser, A. K. El-Naggar, and J. N. Myers Dual Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor Inhibition with NVP-AEE788 for the Treatment of Aggressive Follicular Thyroid Cancer. Clin. Cancer Res., June 1, 2006; 12(11): 3425 - 3434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kim, C. N. Prichard, M. N. Younes, Y. D. Yazici, S. A. Jasser, B. N. Bekele, and J. N. Myers Cetuximab and Irinotecan Interact Synergistically to Inhibit the Growth of Orthotopic Anaplastic Thyroid Carcinoma Xenografts in Nude Mice Clin. Cancer Res., January 15, 2006; 12(2): 600 - 607. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Cell Growth & Differentiation |
