Migration assay

CAF.ERα(+) or CAF.ERα(−) were cultured in 24-well plates. After 24 h, macrophages were seeded on the inserted transwells. After 24 h co-incubation, transwells were washed with PBS and then fixed by 75 % ethanol. Next, transwell membranes were stained with 1 % toluidine blue (w/v, prepared in PBS) and non-migrated macrophages, remaining on the inner transwell surface, were wiped off. Macrophages that migrated to the bottom side of membranes were counted in ten representative areas via microscope (x100 fold).

Conditioned media (CM) collected from the CAF/macrophages co-culture system were used to attract PCa cells invasion via matrigel coated (0.2 mg/ml, 100 μl, air dried overnight) transwells. For the co-culture system, CAF.ERα(+) or CAF.ERα(−) were seeded in the bottom wells of 6-well plates and macrophages were added into the top transwells (pore size is 0.4 μm). The CM was collected from bottom wells after 48 h co-culture. Then, CM was added into each well of new 24-well plates, then matrigel coated transwells were inserted and PCa cells (TRAMP-C1, CWR22Rv1, C4-2, or PC-3, as in figures) at 5 × 10⁴/150 μl were seeded on each transwell. After 24 h incubation, transwells were washed, fixed, and stained. The method for counting invaded cell numbers was the same as with migration assay.

IHC staining was carried out as described previously [64]. Sections were incubated with the primary antibodies, anti-F4/80 (anti-mouse macrophages, Biolegend, San Diego, CA), anti-CD206 (anti-M2 macrophage; sc-20150, Santa Cruz, Dallas, TX), anti-CCL5 (Ameritech Biomedicines, Houston, TX), anti-IL6 (Abcam, ab6672, Cambridge, MA) and anti-firefly (Santa Cruz, Dallas, TX), in 3 % BSA in PBS overnight at 4 °C followed by respective secondary antibodies.

Total RNA was extracted by Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNAs (1 μg) were subjected to reverse transcription using Superscript III transcriptase (Invitrogen). The obtained cDNAs were applied for qPCR using a SYBR green Bio-Rad CFX96 system. Primers used are listed in Table 1. Gene mRNA expression levels were normalized to the mRNA level of GAPDH.

Sequence of qPCR primers

CCL5 promoter luciferase activity was performed using Lipofectamin 2000 (Invitrogen). CAF cells were transfected with CCL5-Luc (0.4 μg) and 1 ng pRL-TK-Luc reporter gene. After transfection, the media were refreshed to 10 % charcoal/dextran stripped (CD)-FBS media for 24 h and 10 nM E2 was added as indicated for an additional 24 h. Cells were then harvested for the dual luciferase assay kit (Promega, Madison, WI).

CM was collected from CAF only or CAF co-cultured with macrophages for ELISA analyses of CCL5 and IL6 (eBioscience, San Diego, CA) according to the manufacturer’s instructions.

For the orthotopic implantation in mice, CWR22Rv-1 cells were transduced with firefly cDNA (22Rv1-Luc). CAF were mixed with 22Rv1-Luc cells (1:9 ratio) and injected into anterior prostate of 8 weeks old athymic nude mice [24, 41]. For cells injection, 22Rv1-Luc/CAF cells (9:1 ratio, total 1 × 10⁶) were suspended in 20 μl of media and Matrigel mix (1:1, v:v). Seven animals were used per group. Mice were monitored by IVIS every 2 weeks for tracking tumor growth and metastasis by intraperitoneal injection of luciferin (Gold Biotechnology, St. Louis, MO) to allow the luciferase to fluoresce. Tumors from primary and metastatic sites were collected after a final IVIS imaging at 12 weeks after implantation. Tumor sizes and macrophages infiltration were compared after IHC staining by macrophage markers. Lymph nodes were stained with firefly luciferase antibody (Santa Cruz, c-12) to confirm cancer cells metastasized from the primary tumor sites. All mice experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Rochester Medical Center.

Values were expressed as mean ± standard deviation (S.D.). The Student’s t test was used to calculate two-sided P values, and considered statistically significant when P < 0.05.