Orthotopic gallbladder cancer modeling

An orthotopic GBC-SD gallbladder cancer-bearing mouse model was established with a high success rate of nearly 90% by implanting minced gallbladder cancer tissue into the left lobe of the liver in BALB/c nude mice. GBC-SD-Luc cells (5 × 10⁶ cells) in 100 μL PBS were subcutaneously injected into the right armpit of each female BALB/c nude mouse. When the tumor volume reached 800 mm³, mice were monitored by bioluminescence using an imaging system (PerkinElmer IVIS® Lumina LT, USA) 10 min after the mice were anesthetized and injected with D-luciferin sodium salt (15 mg mL⁻¹ in 1 × DPBS) at 150 mg kg⁻¹. A mouse tumor with substantial fluorescence intensity was selected, diced into 0.5 mm × 0.5 mm × 0.5 mm pieces, and washed with PBS for orthotopic inoculation preparation. Subsequently, other female BALB/c nude mice were operated on with sterilized instruments, and aseptic principles were maintained. Under the xiphoid process of the mouse, a 1 cm incision was created, and the left liver lobe was extruded via the abdominal orifice. Using the tip of the ophthalmic forceps (XinGong, Chian), the liver leaf was removed by creating a 3mm-long and 3mm-deep channel on its surface. To stop the bleeding, apply pressure to the wound using the sterile cotton swab. The prepared tumor fragment was then placed into the channel on the surface of the liver, and the small opening was compressed for 1 to 2 min using a cotton swab. After the bleeding is stopped, the left lobe of the liver was carefully reinserted into the abdominal cavity, and the abdominal opening was closed layer by layer. As previously mentioned, all mice were monitored with bioluminescence 10 days after surgery. All mice exhibiting detectable fluorescence intensity in the abdomen were regarded as successful models. The mouse was euthanatized when its tumor exceeded the pre-specified maximal tumor volume of 1 cm³ or when its maximum weight loss exceeded 20%, as required by animal ethics.