**3.1 General principles of establishing a NPC animal model**

An important approach in studying the etiology and pathogenesis of malignant tumors is to establish various animal models. As it is impossible to carry out experiments directly on patients, a simple method to induce tumor in animals with high incidence provides a valuable research tool to simulate the human cancer. The following is a discussion on the general principles of establishing a NPC animal model.

#### **3.1.1 Animal selection**

There is a great variation in the susceptibility of different animal species to carcinogens. It is better to choose animals with a low incidence of spontaneous tumors and a predicted high incidence of induced tumor. Some scholars originally believed that animal nasopharyngeal epithelium was not susceptible to aromatic hydrocarbons. Nonetheless, Wang successfully induced experimental "NPC in situ" in mice [51]. However, because rat nasopharynx is analogous to that of humans and rats are readily available, the rat is the preferred model animal. The rats used in these experiments were of mixed breeds, but they very rarely suffered from spontaneous tumors.

As a preferred model animal, the anatomical and histological characteristics of rat nasopharyngeal organs were studied in detail. The anatomical location of rat nasopharynx has been clearly defined [52], but some confusing terms are used in these reports to describe the same structure. For example, rat's nasopharynx is tubular, and some authors called it a nasopharyngeal duct. Actually, rat's nasopharynx is histologically similar to that of humans; both are lined with two kinds of epithelia, stratified squamous epithelium and pseudostratified ciliated columnar epithelium, and have orifices of Eustachian tubes near otopharyngeal end. The term "nasopharyngeal duct" may be confused with another tubular structure lying above the hard palate between the nasopharynx and posterior naris, which is lined completely with ciliated columnar epithelium. This structure is often called the nasopharyngeal tube, and is in fact in the posterior part of nasal cavity. Therefore, if the induced tumor was located above the hard palate, it should be classified as a nasal tumor rather than a nasopharyngeal tumor. Certain terms used in these reports, such as "cancer of nasal cavities," "cancer of nasal turbinates," "ethmoid cancer," and "cancer of nasal sinuses," actually describe malignant tumors developed from nasal turbinates, which are often different from NPC in histological appearance.

Leaton-Jones P et al [52] described histology of normal rat nasopharyngeal epithelium in 1971. Subsequently, Albin N et al [53] have systematically studied serial sections of rat nasopharynxes at different ages. They demonstrated that nasopharyngeal epithelium consists of three different kinds of epithelium: pseudostratified ciliated columnar epithelium, stratified squamous epithelium, and transitional epithelium. In adult rats approximately two-thirds of nasopharynx towards cephalic end is lined with ciliated columnar epithelium. The third near oropharyngeal end is a mixed-type epithelium, with increasing abundance of stratified squamous epithelium towards oropharyngeal end. The cover epithelium on roof and lateral sides of nasopharynx is mainly ciliated columnar epithelial cells, with predominantly continuous stratified squamous epithelium at bottom. The nasopharynx of newborn rats is mainly lined with ciliated columnar epithelium, which

An important approach in studying the etiology and pathogenesis of malignant tumors is to establish various animal models. As it is impossible to carry out experiments directly on patients, a simple method to induce tumor in animals with high incidence provides a valuable research tool to simulate the human cancer. The following is a discussion on the

There is a great variation in the susceptibility of different animal species to carcinogens. It is better to choose animals with a low incidence of spontaneous tumors and a predicted high incidence of induced tumor. Some scholars originally believed that animal nasopharyngeal epithelium was not susceptible to aromatic hydrocarbons. Nonetheless, Wang successfully induced experimental "NPC in situ" in mice [51]. However, because rat nasopharynx is analogous to that of humans and rats are readily available, the rat is the preferred model animal. The rats used in these experiments were of mixed breeds, but they very rarely

As a preferred model animal, the anatomical and histological characteristics of rat nasopharyngeal organs were studied in detail. The anatomical location of rat nasopharynx has been clearly defined [52], but some confusing terms are used in these reports to describe the same structure. For example, rat's nasopharynx is tubular, and some authors called it a nasopharyngeal duct. Actually, rat's nasopharynx is histologically similar to that of humans; both are lined with two kinds of epithelia, stratified squamous epithelium and pseudostratified ciliated columnar epithelium, and have orifices of Eustachian tubes near otopharyngeal end. The term "nasopharyngeal duct" may be confused with another tubular structure lying above the hard palate between the nasopharynx and posterior naris, which is lined completely with ciliated columnar epithelium. This structure is often called the nasopharyngeal tube, and is in fact in the posterior part of nasal cavity. Therefore, if the induced tumor was located above the hard palate, it should be classified as a nasal tumor rather than a nasopharyngeal tumor. Certain terms used in these reports, such as "cancer of nasal cavities," "cancer of nasal turbinates," "ethmoid cancer," and "cancer of nasal sinuses," actually describe malignant tumors developed from nasal turbinates, which are

Leaton-Jones P et al [52] described histology of normal rat nasopharyngeal epithelium in 1971. Subsequently, Albin N et al [53] have systematically studied serial sections of rat nasopharynxes at different ages. They demonstrated that nasopharyngeal epithelium consists of three different kinds of epithelium: pseudostratified ciliated columnar epithelium, stratified squamous epithelium, and transitional epithelium. In adult rats approximately two-thirds of nasopharynx towards cephalic end is lined with ciliated columnar epithelium. The third near oropharyngeal end is a mixed-type epithelium, with increasing abundance of stratified squamous epithelium towards oropharyngeal end. The cover epithelium on roof and lateral sides of nasopharynx is mainly ciliated columnar epithelial cells, with predominantly continuous stratified squamous epithelium at bottom. The nasopharynx of newborn rats is mainly lined with ciliated columnar epithelium, which

**3. Establishment of an animal model of NPC with chemical carcinogens** 

**3.1 General principles of establishing a NPC animal model** 

general principles of establishing a NPC animal model.

often different from NPC in histological appearance.

**3.1.1 Animal selection** 

suffered from spontaneous tumors.

is poorly differentiated. Stratified squamous epithelium appears 10 days after birth. The nasopharyngeal epithelium of 60-day-old rats is similar to that of adult rats.

#### **3.1.2 Selection of carcinogens and induction methods**

Generally, potent carcinogen with short induction time is chosen as tumor-inducing agent, for example, benzo(a)pyrene for lung cancer induction [54] and aflatoxin for liver cancer [55]. Human NPC is too complicated to determine one or two factors because there are many factors involved in NPC development, such as viral and genetic factors, chemical carcinogens in the environment [56]. Moreover, the etiology of human cancers can be complicated, and might not be attributed to a single factor or initiation event [2]. The combined action of chemical carcinogens, viruses, and genetic factor, and also cocarcinogens should be discussed.
