2.7. Animal Study Using Cell Lines and PDX (Patient-Derived Xenograft) Models for Cervical Cancer

To establish the SiHa cell line xenograft tumor, female BALB/c nude mice were purchased from Orient Bio (Seongnam, South Korea). Autoclaved water and food were available to the mice ad libitum. The same SiHa cell line that was used for in vitro experiments was employed. It was cultured in MEM containing 10% FBS. A total of 2 × 10⁶ cells were inoculated subcutaneously in 200 µL of Hanks’ Balanced Salt Solution (HBSS, Biocompare, San Francisco, CA, USA) into the flank of the animals bilaterally. Tumor growth was measured twice a week. The volume of tumors was calculated using a standard formula (length × width² × 0.52), and growth curves were drawn.

To establish PDX models of cervical cancer, surgically removed patient tumor specimens were reduced to small pieces (less than 2–3 mm), implanted into the subrenal capsules of the left kidneys of BALB/c nude mice, and propagated by serial transplantation [11,12]. The clinical information of the patients is provided in Supplementary Table S1 and Supplementary Figures S1–S5. The mice used in these experiments were 6 to 8-weeks old. TAO (8 mg/kg) or PBS was intraperitoneally injected into the model mice once a week for the subsequent 3–4 weeks. For combination therapy with cisplatin, the model mice were either given intraperitoneal cisplatin (4 mg/kg) once a week alone or cisplatin (same dosage) in addition to TAO injections. The mice were then sacrificed and tumors were imaged and weighed. The mice (n = 10 per group) were monitored daily for tumor development and sacrificed when any appeared moribund. We recorded the body weight, tumor weight, and number of tumor nodules. Tumors were fixed in formalin and embedded in paraffin or snap-frozen in optimal cutting temperature (OCT) compound (Sakura Finetek Japan, Tokyo, Japan) in liquid nitrogen. This study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Samsung Biomedical Research Institute (protocol number H-A9-003). The IACUC is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) and abides by the guidelines of the Institute of Laboratory Animal Resources (ILAR). Study of the PDX model for cervical cancers was approved by the Samsung Medical Center Institutional Review Board (IRB file number 2010-04-004) and experiments were performed in accordance with the approved guidelines and regulations.