In vivo cell line derived xenograft (CDX) experiments

Six to eight weeks old female NOD/SCID/γ mice (Jackson lab), housed in pathogen free conventional cages on a 12 hr light-12 hr dark cycle, fed ad libitum were used for tumorigenicity assays. All procedures for mice experimentation were approved by the University of Newcastle Animal Care and Ethics Committee. Luciferase labelled Ishikawa and MFE-296 cells (1.5 × 10⁶) in 200 μL of 1:1 sterile DPBS and Matrigel were injected intraperitoneal (i.p.) into lower abdomen region of female NOD/SCID/γ mice (5/group). Mice were injected i.p. thrice per week with SB-431542 (100 μg/kg), carboplatin (15 mg/kg) and paclitaxel (5 mg/kg) alone or in combination. The endometrial cancer cells were genetically engineered to express the firefly luciferase gene to quantitatively track in vivo growth and metastatic potential. Tumour development and metastatic spread were assessed weekly by monitoring luciferase signal using an IVIS bioluminescent imaging system (PerkinElmer) with a standard protocol over a period of 21 days. Animal health was monitored by daily observation and weekly assessment of weight. After 4 weeks, mice were euthanized and metastatic spread of cancer cells was assessed by counting the number of tumour nodules within the peritoneal cavity of each mouse. Recovered tumours from primary and metastasized sites were fixed in formalin, embedded in paraffin and sections stained with haematoxylin and eosin (H&E) and against antibodies to analyse tumour pathology.