*2.8. Animal Studies*

All animals were maintained in a specific pathogen-free environment. Mie University's Committee on Animal Investigations approved the experimental protocol. Male athymic nude mice (BALB/c, nu/nu, 6–8 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan) and used for all experiments.

### *2.9. In Vivo Xenograft Model*

Examination of the effects of ADT on tumorigenesis of E9, F10, and AIDL cells was performed as previously described [17]. Subconfluent cultures of E9, F10, AIDL, and pcPrF-M5 cells were trypsinized and counted. Xenografts without pcPrF-M5 cells contained 5 × 10<sup>5</sup> PCa cells. Xenografts with pcPrF-M5 cells were prepared by mixing 2.5 × 10<sup>5</sup> PCa cells and 2.5 × 10<sup>5</sup> pcPrF-M5 cells in suspension. Pelleted cells were resuspended in 50 μL neutralized type I rat tail collagen gels and then grafted beneath the renal capsule of male athymic mice (6–8 weeks old). In total, 1 × 10<sup>6</sup> PCa cells were grafted in each mouse. For the androgen deprivation experiments, mice were randomized on day 14 after transplantation. Mice treated with ADT were castrated and orally administered a bicalutamide (5 mg/kg/day) suspension with 0.5% carboxymethylcellulose in a 5-days-on/2-days-off schedule through a 22-gauge gavage needle; the control group underwent sham operation and received the diluent. Mice were killed on days 14 and 21 after ADT, and tumor weights and serum PSA levels were then measured. The tumor volume was determined by direct measurement with calipers (volume = long axis × short axis ×short axis × 0.5236), as previously described [27].
