Fig. 2.
Adding functional AR to PC3 cells (PC3-AR9) decreased prostate cancer cell invasion in bone lesion test and in preclinical animal models. (A) PC3-AR9 cells formed fewer osteolytic lesions than PC3-v cells. Osteoclasts and osteoclast precursors (OC) were cultured with PC3-v and PC3-AR9 cells on cortical bone wafers. After 10 days, the bone wafers were scraped, dried, and stained with the OC cell indicator tartrate-resistant acid phosphatase (TRAP). The extent of bone resorption with PC3-AR9 cells decreased compared to PC3-v cells as shown by measurement of the area of osteoclast lacunae on the bone wafers. OC alone and OC with parathyroid hormone (PTH) were used as the negative and positive control, respectively. Data were from three independent experiments and are presented as mean ± SD *P < 0.05, **P < 0.01. (B, C) PC3-AR9 cells had less bone invasion compared to PC3-v cells. Effects of intratibial injection of PC3-v and PC3-AR9 cells in nude mice. PC3-v cells developed larger osteolytic lesions than PC3-AR9 cells at 6–8 wks (B, representative x-ray radiograph shown), and larger and more invasive tumors as measured by dial caliper at week 12 (C, arrows and quantitated tumor volume, Lower). Data are shown as mean ± SD; *, P < 0.05; **, P < 0.01. (D) PC3-AR9 cells generated smaller metastatic PLN tumors compared to those generated by PC3-v cells. PC3-v and PC3-AR9 cells (5 × 105) suspended in 100 μl Matrigel were inoculated into the anterior prostate of 16-wk-old nude mice. Twelve weeks after injection, large PC3 prostate tumors developed (not shown). To visualize PLN-metastatic tumors, we removed prostates containing primary tumors. Note that larger PLN-metastatic tumors (shown by gross and H&E staining, Left) were developed in mice inoculated with PC3-v tumors (arrows). The tumor volume was quantitated (right panel, n = 5). (E) PC3-AR9 cells recombined with WPMY1-v or WPMY1-ARsi cells generated smaller metastatic tumors than their control PC3-v recombined groups (arrows). PC3-v or PC3-AR9 cells (5 × 105), combined with WPMY1-v or WPMY1-ARsi cells, were suspended in 100 μl of Matrigel and inoculated into the anterior prostate of 16-wk-old nude mice. Twelve weeks after injection, the tumors developed (not shown), and the sizes of PLN metastatic tumors were compared via gross appearance and histology (H&E). The tumor volume was quantitated (Right, n = 5).