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. 2013 Jun 1;6(3):273–281. doi: 10.1593/tlo.13175

The Histone Methyltransferase EZH2 Mediates Tumor Progression on the Chick Chorioallantoic Membrane Assay, a Novel Model of Head and Neck Squamous Cell Carcinoma1

Min Liu *,2, Christina Springstead Scanlon *,2, Rajat Banerjee *, Nickole Russo *, Ronald C Inglehart *, Amanda L Willis †, Stephen J Weiss †, Nisha J D'Silva *,‡
PMCID: PMC3660795  PMID: 23730406

Abstract

Current in vivo models for head and neck squamous cell carcinoma (HNSCC) have limitations in simulating some essential tumorigenic phenotypes, such as invasion. Most mouse models of human HNSCC are inadequate because tumor cells are injected directly into the connective tissue, thereby bypassing the basement membrane of the surface epithelium, the first barrier to invasion. In this manuscript, we establish the chick chorioallantoic membrane (CAM) assay as an in vivomodel of human HNSCC tumor progression. Using the CAM model of HNSCC, we investigated the role of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, in multiple aspects of HNSCC tumor progression. We found that knockdown of EZH2 reduced tumor size, angiogenesis, invasion, and metastasis of tumors produced by grafting human HNSCC cells onto the CAM. In addition, we demonstrate that EZH2 expression mediates a mesenchymal phenotype in HNSCC cell lines and mouse tumors. These findings demonstrate the advantages of the newly proposed CAM model of human HNSCC and highlight the emerging role of EZH2 in HSNCC tumor progression.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally [1]. The 5-year survival rate is about 50%, which is poorer than breast cancer or melanoma [2]. New treatments are required since current regimens have not improved survival in over five decades. Understanding the mechanisms that control HNSCC progression will provide new strategies for tumor eradication. Current in vivo models for human HNSCC do not simulate invasion, an essential tumorigenic phenotype, which may explain the weak correlation between successful preclinical studies and clinical success with the same antagonist.

The tumorigenic phenotypes or hallmarks of cancer include proliferation, survival, invasion and metastasis, angiogenesis, and stemness [3]. Cancer cell proliferation and survival promote tumor growth. Invasion is required for multiple steps in HNSCC progression including initiation, local spread, and metastasis. During transformation of a precancerous lesion to HNSCC, cells invade from the surface epithelium, the tissue of origin of HNSCC, into the underlying connective tissue. Invading cells destroy the basement membrane that separates the epithelium from the connective tissue. Destruction of the basement membrane and invasion are essential for development of HNSCC. Thus, the basement membrane is the first, most robust structural barrier to invasion [4]. Angiogenesis facilitates tumor growth and spread, and stemness promotes tumor recurrence. Given the importance of these phenotypes in tumor progression, a robust cancer model should recapitulate these phenotypes.

Many models have been developed in the last few decades to assess the oncogenic phenotypes of HNSCC. However, most of these models are in vitro systems that work with monolayer cultures, making these assays difficult to translate into clinical application. Recently, we developed an in vitro three-dimensional model for human HNSCC [5], but this oral cancer equivalent model does not simulate the systemic impact of invasion in vivo. Most mouse models of human HNSCC are inadequate because tumor cells are injected directly into the connective tissue, thereby bypassing the basement membrane of the surface epithelium, the first barrier to invasion. Given the importance of invasion in tumor progression, we developed a novel in vivo model of invasion of human HNSCC using the chicken embryo model.

The chicken embryo model has developed into the cornerstone of cancer biology over many decades. This system has long served as the principal model system for developmental biology and has provided a pathway for critical conceptual development in genetics, immunology, virology, and cancer biology. The developing chicken egg attracted interest from some of the earliest known scientific investigations dating back to ancient Egypt and Greece [6]. The wide accessibility of chicken eggs has helped to maintain the popularity of the model for thousands of years. The chicken egg model has essentially contributed to the most significant scientific discoveries of many Nobel laureates. For example, the causal link between viruses and cancer [7], the first known oncogene [8], the mechanism of reverse transcriptase and RNA viruses [9], and the discovery of neural growth factor [10] are among the many key scientific findings empowered by the chicken embryo system.

As early as 1913, scientists discovered that tumor grafts can be cultivated by the rich capillary plexus of the chick chorioallantoic membrane (CAM) surrounding the chicken embryo [11]. The CAM model was developed as a model of angiogenesis in the 1970s [12], and by the 1980s, it was identified as a tool to study tumor metastasis [13]. These early cancer studies using the chicken embryo system paved the way for recently developed methods of studying invasion in cancer using the CAM model.

Although the CAM assay has been known for many years, the benefits of studying tumor invasion using this model are more recently recognized. The CAM is a highly vascularized membrane that is located directly below the eggshell. This makes the CAM easy to access through a small hole in the eggshell. The CAM is also made up primarily of type IV collagen, which simulates the basement membrane of human oral epithelium. The CAM assay has been used to measure invasion of a variety of cell types, including fibroblasts [14] and several types of cancer cells, including melanoma cells [15–17].

We propose that the chick embryo is an excellent model of invasion and metastasis of human HNSCC. The CAM consists of the chorionic epithelium separated from the underlying allantoic membrane by connective tissue. The chorionic epithelium is separated from the connective tissue by an epithelial-derived basement membrane that contains type IV collagen [14]. The cellular connective tissue contains type I and III collagen and blood vessels. In this model, HNSCC cells are seeded on top of the CAM and allowed to invade. Thus, the CAM recapitulates intraoral human HNSCC progression including disruption of the basement membrane, complexity of the connective tissue, angiogenesis, and metastasis. Even the histopathologic features simulate invasion observed in HNSCC. Destruction of the basement membrane can be easily visualized, and tumor growth, invasion into the connective tissue, and metastasis can be accurately quantified, making this a valuable model for investigating progression of HNSCC.

In this study, we describe for the first time the use of the CAM to investigate multiple tumorigenic phenotypes, including tumor growth, invasion, metastasis, and angiogenesis in HNSCC. Recently, we showed that enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, promotes progression of HNSCC by inducing multiple cancer phenotypes, likely through methylation of multiple tumor suppressor gene [18,19]. Using the CAM model, we investigated the role of EZH2 in tumor growth, angiogenesis, invasion, and metastasis in vivo. In addition, we show the role of EZH2 in epithelial-mesenchymal transition (EMT) in mouse tumors, CAM tumors, and HNSCC cell lines. Overall, we are able to establish the CAM model of HNSCC and investigate the role of EZH2 in several hallmarks of tumor progression.

Materials and Methods

Cell Culture

The HNSCC cell line, UM-SCC-29 (from Thomas Carey, University of Michigan) used in this study was validated by genotyping at the University of Michigan DNA Sequencing Core and cultured as described [20,21]. Cells were maintained in Dulbecco's modified Eagle's medium (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% FBS and 1% penicillin/streptomycin. EZH2 in HNSCC cells was stably downregulated as described [15]; scrambled shRNA (shSCR) was used for control cells and shEZH2 for EZH2 knockdown cells (Open Biosystems, Huntsville, AL). Cells were selected with puromycin (Sigma-Aldrich Corp, St Louis, MO).

Immunoblot Analysis

Immunoblot analysis was performed as previously described [22]. NP-40 lysis buffer (1%) was used to lyse HNSCC cells. EZH2 (BD Biosciences, San Jose, CA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Millipore, Billerica, MA) primary antibodies were used. The secondary antibody used was HRP-conjugated anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA). SuperSignal West Pico Chemiluminescent System (Thermo Scientific, Rockford, IL) was use to visualize immunoreactive proteins, and ImageJ software was used to quantify signal intensity (http://rsbweb.nih.gov/ij/).

Immunohistochemistry

Immunohistochemistry of tissue sections was performed as described [21]. The primary antibodies used were vimentin (Proteintech, Chicago, IL) and E-cadherin (BD Biosciences), and biotinylated goat anti-rabbit and biotinylated goat anti-mouse secondary antibodies were used (Biocare Medical, Concord, CA). Imaging of cells was performed at the Microscopy and Image Analysis Core at the University of Michigan on an Olympus BX-51 microscope. Representative fields were imaged at x20.

Murine Model of HNSCC Using Subcutaneous Injection

Athymic nude mice were used as described [23]. UM-SCC-29 cells (1 x 106) were stably transduced with shSCR or shEZH2 and injected subcutaneously to assess tumor growth [15]. Histopathologic analysis of these tumors is shown in the present study.

Immunofluorescence Detection of Vimentin and E-Cadherin

Cells were labeled with vimentin (Proteintech) and E-cadherin (BD Biosciences) primary antibodies diluted in 0.3% Triton X-100 overnight at 4°C, washed, and incubated for 2 hours at room temperature in an appropriate conjugated secondary antibody, washed, and incubated in 4′,6-diamidino-2-phenylindole (DAPI; 1:3000) for 3 minutes. Imaging of cells was performed at the Microscopy and Image Analysis Core at the University of Michigan on an Olympus BX-51 microscope. Representative fields were imaged at x100.

Data Analysis

Statistical analysis was performed using Student's t test with GraphPad Prism (GraphPad Software, San Diego, CA). A P value of <.05 was accepted as statistically significant.

Results

CAM In Vivo Model of HNSCC Tumor Progression

Figure 1 provides an overview perspective of the chicken embryo and identifies where HNSCC tumors grow, invade, and metastasize within the developing egg. HNSCC cells are seeded on the upper CAM and destroy the basement membrane of the surface epithelium to invade the connective tissue and blood vessels through which they metastasize to the lower CAM and liver. Figure 2 provides an overview of the procedure and outlines the end-point assays including tumor growth, invasion, angiogenesis, and metastasis. The University of Michigan Unit for Laboratory Animal Medicine was consulted regarding ethical use of the chicken embryo CAM for experiments.

Figure 1.

Figure 1

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Overview of the CAM model of tumor progression. Fluorescently labeled cancer cells are seeded on the upper CAM of the chick embryo. The cancer cells invade the epithelium and basement membrane of the upper CAM and move through connective tissue into the vasculature. Cancer cells can metastasize to the lower CAM or liver and lung of the developing chicken.

Figure 2.

Figure 2

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Experimental procedure and time line of the CAM assay. Fertilized chicken eggs are hatched at day 0. On day 8, the developing vasculature is identified and a window is opened on the egg to seed human cancer cells. On day 11, the egg can be reopened to harvest the upper CAM containing the tumor to assess tumor growth, invasion, and angiogenesis. For metastasis studies, the egg is opened at day 16 to collect the lower CAM and liver of the developing chicken. Quantitative PCR analysis of the collected tissues provides an estimation of the number of human cancer cells that have invaded to the collected organs.

Dropping the CAM. Fertilized commercial Lohmann White Leghorn eggs were obtained from the Michigan State University Department of Animal Sciences Poultry Farm. Before hatching, the eggs were maintained at 24°C. Eggs were subsequently “hatched” in a humidified incubator (Digital Sportsman Incubator; G.Q.F. Manufacturing, Savannah, GA) at 38°C with 60% humidity. The initial day of incubation is considered day 0. On day 11, the following structures were labeled on the egg using the ACE light source (Trevigen Inc, Gaithersburg, MD): large blood vessel, umbilical cord, air sac, small square window for the artificial air sac generation, and large window area for seeding cancer cells. Using a Dremel 1100-N/25 7.2-Volt Stylus Lithium-Ion Cordless Rota (Robert Bosch Tool Company, Stuttgart, Germany), a 1-cm2 window was drilled on the top of the eggshell, maintaining the outer eggshell membrane. A pinpoint hole was prepared on the side of the egg at the location of the air sac. Hank's balanced salt solution (HBSS, 25 µl; Invitrogen, Life Technologies, Grand Island, NY) was added on top of the 1-cm2 window at the top of the egg. Then, using a 30.5-gauge needle, the outer eggshell membrane was punctured at the location of the window so that the buffer separated the outer eggshell membrane from the CAM. The small pinpoint hole was vacuumed using a Pasteur pipette bulb, causing the air bubble to move to the window and allowing the CAM to drop. Then, the eggshell was drilled in the large window area, and blunt-ended forceps were used to peel off the eggshell membrane without disturbing the CAM. The large square hole was covered with parafilm and the egg was place in the incubator without shaking.

Seeding HNSCC cells. HNSCC cells (1 x 106) were resuspended in 5 µl of HBSS. The pipette was used to make a bead of cell and medium that is dropped onto the CAM surface, without allowing the pipette tip to touch the CAM. The square window on the egg was sealed with Tegaderm HP Transparent Film Dressing. The eggs were incubated without shaking for approximately 3 days.

Harvesting the CAM. Using a needle and syringe, a small amount of 4% paraformaldehyde was injected onto the surface of the CAM. Dissecting scissors were used to cut open the large window to visualize the CAM and the tumor. Using scissors and a pair of forceps, each CAM was lifted and cut around the tumor. The tumors and surrounding CAM were transferred to a six-well dish containing 4% paraformaldehyde and incubated at 4°C for 4 hours. The CAMs were transferred to cold 30% sucrose and stored overnight at 4°C. The next day, the CAMs were embedded in Optimal Cutting Temperature Compound (Tissue-Tek, Sakura Finetek USA, Torrance, CA) and frozen at -80°C until sectioning and staining. Tissue sections (8–10 µm) were fixed in 4% paraformaldehyde for 5 minutes followed by staining with hematoxylin and eosin.

End-point assays. For tumor growth and angiogenesis studies, suspensions of 5 x 105 HNSCC cells with stable knockdown of EZH2 or controls were suspended in 5 µl of HBSS and plated on the upper CAM. The window on the eggshell was resealed with adhesive tape and eggs were returned to the incubator for 48 hours before harvesting the tumor (n = 5 chick embryos per experimental group). Surface area of the tumors was quantified using ImageJ software and statistically compared between control tumors and tumors with stable EZH2 knockdown. Angiogenesis was also quantified using red color density within 200 µm of tumors in images using ImageJ software (http://rsbweb.nih.gov/ij/) with the Colour Threshold plugin provided through the University of Birmingham School of Dentistry web site (http://www.dentistry.bham.ac.uk/landinig/software/software.html).

For invasion assays, cells were dyed with the lipophilic tracer, DiO (a dialkylcarbocyanine derivative) before experiments. Tumor sections were imaged at x20, and invasive islands were quantified for each image. Statistical analysis to compare the number of invasive islands was performed.

For type IV collagen staining, the frozen tissue sections were fixed in methanol, washed in phosphate-buffered saline (PBS), and blocked with 0.1% BSA and 10% normal goat serum (NGS) in phosphate-buffered saline for 30 to 60 minutes. The type IV collagen antibody was diluted 1:1 in 0.1% BSA and 5% NGS and incubated on the tissue sections for 2 hours. The coverslips were mounted with Prolong Gold Antifade Reagent with DAPI (Invitrogen, Life Technologies).

For metastasis experiments, HNSCC cells were plated as described for invasion studies at day 8. The lower CAM, liver, and lungs were collected at day 16. Human DNA was quantified from DNA extracted from the harvested tissues using Alu-polymerase chain reaction (PCR) to compare metastasis from control and EZH2 knockdown tumors. To generate the standard curve, genomic DNA from human HNSCC cells (each human cell contains 6.6 pg of DNA) was mixed with 1 µg of chicken genomic DNA in logarithmically increasing concentrations as 0.1, 1.0, 10, 100, 1000, and 10000 cells. PCR was performed in triplicate for each of the standards as well as the experimental samples. The absolute number of metastatic human cells in the experimental sample was calculated from the standard curve using linear regression.

EZH2 Enhances HNSCC Tumor Size on the CAM

Using the CAM in vivo model, we investigated the impact of EZH2 on tumor growth in HNSCC. EZH2 is a master regulatory gene in HNSCC that inhibits expression of tumor suppressor genes [18]. UM-SCC-29 cells with stable knockdown of EZH2 (UM-SCC-29-shEZH2) and corresponding control cells with empty vector (UM-SCC-29-shSCR) were seeded on the CAM (n = 5 for each group). After 48 hours, the upper CAM was harvested from each chick embryo and the surface area of the tumors was quantified (Figure 3A). shRNA-mediated EZH2 knockdown was confirmed by immunoblot (Figure 3B). UM-SCC-29-shEZH2 cells produced tumors that were significantly smaller than tumors generated by control cells (P = .0460; Figure 3, A, dashed lines, and C). Although the difference in size between control and EZH2-deficient tumors is significant, the variability in tumor size led to a higher P value than anticipated. A larger sample size may have provided a lower P value to better reflect the difference in tumor size between the groups. Additionally, because some tumors appear to be more bulky than others, three-dimensional analysis of tumor size may provide a more consistent estimation of tumor size.

Figure 3.

Figure 3

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EZH2 promotes tumor growth and angiogenesis. Fluorescently labeled UM-SCC-29-SCR and UM-SCC-29-shEZH2 cells were seeded on the CAM. The upper CAM and tumors were collected 2 days later to analyze tumor growth and angiogenesis. White arrowheads show blood vessel growth approximating tumor (A, bright-field images). Dashed lines outline tumor generated from HNSCC cells on CAM [A, green fluorescent protein (GFP) images]. Yellow arrowheads identify tumor islands migrating from primary tumor (A, GFP images). shRNA-mediated EZH2 knockdown was confirmed by immunoblot (B). The average tumor growth and blood vessel density were calculated for both shSCR and shEZH2 tumors (n = 5 for each group). Tumor size (C) and angiogenesis (D) were significantly decreased for shEZH2 tumors compared to control tumors.

EZH2 Promotes Angiogenesis of HNSCC Tumors on the CAM

To investigate the impact of EZH2 on tumor-associated angiogenesis, the area of blood vessels within 200 µm of the tumors were quantified. shEZH2 tumors had decreased blood vessel area adjacent to tumors compared to controls, indicating decreased angiogenesis of the tumors (P = .0348; Figure 3, A, arrows, and D).

EZH2 Enhances Basement Membrane Disruption and Invasion of HNSCC Tumors on the CAM

Tumors produced by UM-SCC-29-shSCR and UM-SCC-29-shEZH2 were harvested and sectioned. Type IV collagen staining was performed on the section to visualize disruption of the basement membrane on the upper CAM. Tumors produced with UM-SCC-29-shSCR control cells showed more disruption of the basement membrane than tumors with stable knockdown of EZH2 (Figure 4A). This correlated with an increased number of invasive tumor islands in UM-SCC-29-shSCR tumors than in tumors from cells with downregulation of EZH2 (P = .0053; Figure 4B).

Figure 4.

Figure 4

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EZH2 promotes destruction of the basement membrane and invasion. Arrows (A) identify the basement membrane structure, and tumor cells are labeled green. shSCR tumor cells are highly proliferative and invasive and destroy the basement membrane structure, but shEZH2 cells do not disrupt the basement membrane. Fewer invasive tumor islands (B) are observed on the histology of shEZH2 tumors (sample 4 shown) than control tumors (sample 3 shown).

EZH2 Promotes a Mesenchymal Phenotype of HNSCC In Vitro and of Murine and CAM Tumors

Previously, we established that down-regulation of EZH2 inhibited tumor growth in mice [18]. Control tumors that express high EZH2 exhibited an aggressive phenotype, and were comprised of cells with large nuclei, little cytoplasm, and spindled morphology (arrows), that invaded skeletal muscle (arrowheads; Figure 5A, upper panel). In contrast, tumors with EZH2 knockdown (Figure 5A, lower panel) exhibited well-differentiated epithelial cells (keratin formation; arrowheads) with increased cytoplasm (arrows), a less aggressive, more epithelioid phenotype. To verify the impact of EZH2 on EMT, UM-SCC-29-shEZH2 and UM-SCC-29-shSCR cells were plated at 60% confluence and fixed. Immunofluorescence labeling of vimentin and E-cadherin were performed, and five representative fields were imaged at x100 (Figure 4B). Intensity of fluorescence was quantified and normalized to the average intensity of shSCR cells. Control cells have a more mesenchymal phenotype with increased vimentin (P < .001) and decreased E-cadherin (P = .0342) compared to cells with EZH2 knockdown (Figure 5B). These findings are consistent with EZH2 inducing an EMT phenotype. In addition, immunohistochemistry was performed for vimentin and E-cadherin expression on CAM tumor sections from shSCR and shEZH2-treated cells (Figure 5C). Tumors with EZH2 knockdown had decreased vimentin expression (arrows) and higher E-cadherin staining (arrows) than control tumors.

Figure 5.

Figure 5

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EZH2 promotes EMT and metastasis of HNSCC. (A) Histopathologic appearance of HNSCC tumors induced by UM-SCC-29-shSCR and UM-SCC-29-shEZH2 cells in mice (A). Control tumors (upper panel) exhibit an aggressive and mesenchymal phenotype with large nuclei, little cytoplasm, and spindled morphology (arrows) and invasion into skeletal muscle (arrowheads). Knockdown of EZH2 (lower panel) leads to more epithelioid, well-differentiated tumors containing cells with increased cytoplasm (arrows) and keratin formation (arrowheads). To verify the impact of EZH2 on EMT, immunofluorescent labeling of vimentin and E-cadherin was performed and representative fields were imaged at x100 (B). Relative fluorescence was measured for five representative fields and quantified. Control cells have a more mesenchymal phenotype with increased vimentin (P < .001) and decreased E-cadherin (P = .0342) compared to cells with EZH2 knockdown. HNSCC cells were seeded on the CAM, and the lower CAM and liver were collected at day 15. Immunohistochemistry of EMT markers on CAM sections shows decreased vimentin (arrows, C) and more intense E-cadherin expression of tumor cells (arrows, C) for shEZH2 tumors compared to shSCR tumors. Metastases were observed for both the lower CAM and liver for controls, but no metastases were observed for any shEZH2 tumors (D and E).

EZH2 Promotes Metastasis of HNSCC Tumor Cells on the CAM

The invasive phenotype of tumor cells facilitates extension into the surrounding structures and spread to distant sites (metastasis) through the blood vessels. In the CAM model, metastasis requires invasion of cells through the basement membrane of the surface epithelium and into the blood vessels. Since EZH2 promotes invasion, we also investigated its effect on metastasis using the CAM model. UM-SCC-29-shSCR and UM-SCC-29-shEZH2 cells were incubated on the CAM of day 7 chick embryos. The eggs were incubated until day 15, when the lower CAM and liver of the developing chick were harvested. The metastasized human cells in the chicken background were quantified as described by quantitative PCR for amplification of human Alu sequences [24,25], which eliminates cross-reactivity with chicken DNA. When using control UM-SCC-29 cells, metastases were detected in all lower CAM specimens and four of five livers of the developing chicks. However, when using cells with reduced EZH2 expression, no metastases were detected in either the lower CAM or liver in any samples (Figure 5, D and E; P = .0151).

Discussion

Over the past decade, the histone methyltransferase EZH2 has emerged as a key player in tumor progression in many cancer types, including HNSCC [18], breast [26,27], bronchial [28], lung [29], and prostate [30]. Overexpression of EZH2 is often linked to poor prognosis and advanced disease [31]. EZH2 expression is also correlated to increased angiogenesis in tumors [32], in part due to paracrine signaling between tumor cells and associated vasculature [33]. EZH2 has also been shown to have a role in cancer stem cell maintenance [34].

Our laboratory recently showed that EZH2 contributes to HNSCC progression by hypermethylating the promoter region of the tumor suppressor Rap1GAP [18,19]. We found that EZH2 is upregulated in HNSCC cell lines compared to normal keratinocytes and that EZH2 promotes tumor growth in vitro and in vivo in a mouse model of HNSCC. In our previous study, we also evaluated the role of EZH2 in invasion using in vitro assays and found that EZH2 expression is highly correlated with HNSCC cell invasion [18]. However, we were unable to evaluate the impact of EZH2 on early invasive phenotypes, i.e., destruction of the basement membrane of surface epithelium, since, in the mouse model, tumor cells are injected directly into the connective tissue. Invasion beyond the basement membrane is required for transformation of a precancerous lesion (epithelial dysplasia) to HNSCC [35].

In our current study, we chose the CAM in vivo model of tumor progression to validate our previous in vitro findings about the role of EZH2 in tumor invasion. In this study, which is, to our knowledge, the first study to describe the use of the CAM model to investigate tumor progression of HNSCC, we show that down-regulation of EZH2 in HNSCC cells inhibits destruction of the basement membrane and decreases invasion in vivo. In addition, we show that EZH2 mediates angiogenesis, growth, and metastasis of HNSCC.

There are many benefits of using the CAM to study tumor progression [36]. The CAM assay is completed in a short time period and is relatively inexpensive compared to most in vivo models. The lack of a mature immune system at the time the assay is performed allows for use of different cell types and cells from different species. Because the chicken embryo has been used scientifically for centuries, the system is well described in the literature. Limitations of the assay include the extensive optimization and the large number of eggs that are required to obtain consistent results. As in other in vivo systems, tumors produced on the CAM exhibit some variability. Therefore, it is appropriate to use a sample size of at least five eggs per group to characterize differences.

In addition to establishing the CAM model of HNSCC tumor progression, we also evaluated the role of EZH2 expression on the histopathologic presentation of HNSCC tumors and on the expression of the EMT markers vimentin and E-cadherin. EMT is a process by which nonmotile cells lose contact with neighboring cells and become more motile [37]. EMT has been shown to promote HNSCC invasion, metastasis, and tumor stemness [35]. While control cells produced aggressive, mesenchymal-like tumors in vivo, tumors produced from HNSCC cells with reduced EZH2 expression had a more epithelial-like appearance, consistent with a less aggressive tumor. In addition, knockdown of EZH2 leads to decreased vimentin and increased E-cadherin expression in HNSCC cells and CAM tumors. These findings indicate that EZH2 plays a role in mediating EMT in the HNSCC cell line UM-SCC-29. We have previously shown that EZH2-mediated invasion is not dependent on E-cadherin alteration in the E-cadherin-deficient cell line, OSCC3. Therefore, other factors that are still under investigation have a role in EZH2-mediated EMT, independent of E-cadherin [19].

Our study investigating the role of EZH2 in tumor progression is the first to describe the use of the CAM model to study progression of HNSCC. The CAM model can be used to investigate tumor size, angiogenesis, invasion, and metastasis of HNSCC. In addition, we show that knockdown of EZH2 expression in HNSCC cells leads to less aggressive tumors with a more epithelial-like phenotype. Together, these studies highlight the emerging role of EZH2 in HNSCC progression. Future studies will elucidate the mechanistic role of EZH2 in EMT. In addition, the role of EZH2 inhibitors should be explored as a therapeutic option for HNSCC treatment.

Acknowledgments

The authors thank Kenneth Rieger for his artistic assistance with illustrations.

Footnotes

1

This work was supported by National Institute of Dental and Craniofacial Research grants DE018512 and DE019513 (N.J.D.) and DE021293 (C.S.S.).

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