In vivo tumorigenesis assay in the murine xenograft model

Severe combined immunodeficiency (SCID) mice were purchased at ages 4 to 6 weeks from the BioLASCO (Taipei, Taiwan) and housed in an isolator and fed with autoclaved food ad libitum. For tumor growth, cancer cells were trypsinized, washed, centrifuged, and resuspended. A total volume of 0.1 mL containing 4×10⁶ cells was subcutaneously injected into the dorsal region of each mouse. All of the mice were evaluated using DCE and DW-MRI at the 2^nd (baseline), 3^rd and 4^th weeks after tumor implantation. After the final MR study, mice were anesthetized with intraperitoneal injection of 1.2% Avertin and then sacrificed by intravenous injection of potassium chloride (2 meq/kg). The tumors were confirmed by histological examinations, and the excised tissues were fixed and embedded in ornithine carbamyl transferase. From each frozen block, three consecutive sections were obtained for hematoxylin and eosin (H&E), microvessel and apoptosis staining. All animal experiments were performed in triplicate and all procedures for animal experimental protocols were approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine.