3. Orthotopic tumor xenograft models

Two methods were used to establish tumor xenograft at the cervical and abdominal part of the esophagus in nude mice. In the first method to establish tumor xenograft at the cervical esophagus, a 10-mm incision was first introduced vertically and through the neck skin of an anesthetized mouse, which was prepared by intraperitoneal injection of sodium pentobarbital at 54 mg/kg. Cervical muscles, salivary glands, and collective tissues between the trachea and the esophagus were carefully separated by fine point forceps, then the trachea was pulled lightly aside by serrated tip forceps to expose part of the cervical esophagus for cell injection. ESCC cells suspension in 20 μL was injected into the esophageal wall using a 29-gauge BD Ultra-Fine insulin syringe (BD Biosciences, San Jose, CA) (Fig. 1A). A small edema at the cell injection site indicated successful cell injection. The cell suspension was prepared by mixing 1×10⁶ ESCC cells in serum-free medium with an equal volume of Matrigel (Corning, Tewksbury, MA). After the surgical procedure, the skin was closed using non-absorbable 5-0 polypropylene suture (Ethicon, Somerville, NJ). In the second method to establish tumor xenograft at the abdominal part of the esophagus, a horizontal incision of 15 mm was made through the skin and muscle right below the rib cage of an anesthetized mouse using dissection scissors. After putting aside the liver and the stomach with serrated tip forceps, part of the abdominal esophagus between stomach and diaphragm was exposed for cell injection. ESCC cell suspension prepared above was then injected into the esophageal wall (Fig. 1B) and the appearance of small edema at the cell injection site indicated successful cell injection. For both methods, body weight and survival time of the mice were monitored after tumor cell injection. At the experimental end-point, the mice were sacrificed, while the tumor xenografts were dissected for size measurement. Serial cross-sections of the esophagus prepared from paraffin-embedded samples were stained with hematoxylin and eosin to detect any tumor invasion events into the esophageal lumen. These sections were also stained with Ki-67 and CD34 following a standard immunohistochemistry protocol. Histological images were viewed under a microscope and captured using a DXM1200F digital camera (Nikon, Tokyo, Japan).

Surgical procedures to inject esophageal squamous cell carcinoma (ESCC) cells at different anatomical locations in the mouse esophagus. ESCC cells mixed with Matrigel were injected into the esophageal wall at the cervical esophagus (A) and at the abdominal esophagus (B).