Xenograft tumor studies

CCA samples were obtained from the EHBH. The human studies were approved by the Ethics Boards of EHBH, and written informed consent was obtained from each patient. Likewise, all animal procedures were approved by the Ethics Board of EHBH. For cell line xenografts, cancer cells (1 × 10⁶) in a phosphate-buffered saline (PBS)/Matrigel mixture (1:100 μl) were injected subcutaneously into 6-week-old female nude mice. When the tumors reached about 100 mm³, the mice were divided randomly into different groups (n ≥ 4). For the PDX model, fresh surgical tumor tissues (P₀) were sectioned into ~3-mm³ pieces and implanted subcutaneously into the flanks of female NOD/SCID or NSG mice. After 2 to 4 months of tumor growth, the established PDX tumors (P₁) were transplanted into subsequent generations (P₂-P_n) under the skin of nude or NOD/SCID mice. When the tumors reached about 100 mm³, the mice were assigned randomly to different groups (n = 5). For PTEN reexpression, purified PTEN or null adenovirus (Ad-PTEN or Ad-null, 2 × 10⁸) was injected intratumorally every 3 days at multiple locations. For bortezomib treatment, the mice received an intraperitoneal dose of bortezomib (0.2 or 0.5 mg/kg). The tumor sizes were measured every 3 days using caliper, and the tumor volumes were calculated using the 0.5 × L × W², in which L is the longest diameter and W is the shortest diameter. Hematoxylin and eosin (H&E) and IHC were performed on paraffin-embedded xenograft tumor specimens after the mice were euthanized by CO₂ asphyxiation when tumors reached 1500 mm³.