Prostate cells’ orthotopic implantation

Our animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals. Male 6- to 8-week-old nude mice were purchased from NCI (Bethesda, MD, USA). The Matrigel (1:1) mixtures with 1 × 10⁶ CWR22Rv1 cells (with stable transfected luciferase) with or without lentiviral stable expression of miR-361-3p were orthotopically injected into both anterior prostates. After 1 week of implantation, the mice with vector or miR-361-3p expression were separated randomly into two groups with matching charateristics and treated with either DMSO or 35 mg/kg Enz by i.p. injection 3×/week for 2 weeks. The separation of two groups was not blinded to the operator. Tumor formation and sizes were monitored weekly during the 2 weeks of drug treatment by the noninvasive In Vivo Imaging System (IVIS). After another week and a final IVIS imaging, mice were sacrificed and tumors were removed. The tissue samples were then fixed and processed as paraffin tissue sections.