*2.2. Cell Culture*

The androgen-sensitive, AR-positive human PCa cell lines LNCaP and 22Rv1 were obtained from the American Type Culture Collection (Manassas, VA, USA). Both LNCaP and 22Rv1 cells were authenticated by the short tandem repeat-PCR method and were cultured in phenol red (+) RPMI-1640 supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich Co., LLC.). E9 and F10 cells (showing low sensitivity to androgen) were obtained from the parental androgen-sensitive LNCaP cells using a limiting dilution method under regular culture conditions [22,23]. In contrast, androgen-insensitive AIDL cells were established from LNCaP cells by continuous passaging under hormone-depleted conditions [24]. AIDL cells were cultured in phenol red (-) RPMI-1640 supplemented with 10% charcoal-stripped (CS)-FBS. The androgen sensitivity of parental LNCaP, E9, F10, and AIDL cells was confirmed by changes in *KLK3* (PSA) mRNA expression in cell cultures treated with synthetic androgen R1881 [19]. The nontumorigenic human prostate epithelial cell line BPH-1 was obtained from Dr. Simon W. Hayward (Northshore University HealthSystem, Chicago, IL, USA) and was cultured in phenol red (+) RPMI-1640 supplemented with 10% FBS.

Commercially available human prostate stromal cells (PrSC) were purchased from Lonza Group Ltd. (Basel, Switzerland). pcPrFs (pcPrF-M5, pcPrF-M6, and pcPrF-M7) were primary cultured from PCa specimens collected from biopsies of patients with advanced PCa [17]. PrSC and pcPrFs were cultured in medium prepared from an SCBM Bullet Kit (Lonza Group Ltd.). The four fibroblast lines (PrSC, pcPrF-M5, pcPrF-M6, and pcPrF-M7) do not express AR protein and do not respond to DHT stimulation on cell proliferation as previously reported [17].

### *2.3. Indirect Coculture of Prostate Cancer Cell Lines (E9, F10, and AIDL Cells) with Fibroblasts*

E9, F10, and AIDL cells were co-cultured with each of the four fibroblast lines (PrSC, pcPrF-M5, pcPrF-M6, and pcPrF-M7) in six-well plates using cell culture inserts (BD Falcon, Franklin Lakes, NJ, USA) as previously described [17]. E9, F10, and AIDL cells (4 × 10<sup>4</sup> cells/well) were seeded into six-well plates in their respective recommended medium, whereas fibroblasts (PrSC, pcPrF-M5, pcPrF-M6, and pcPrF-M7; 2 × 10<sup>4</sup> cells/well) were seeded in SCBM media into cell culture inserts for 2 days. The culture medium for PCa cells and fibroblasts was replaced with phenol red (-) RPMI-1640 supplemented with 1% CS-FBS containing DHT (0.1 nM), and the inserts with fibroblasts were then placed into six-well plates for an additional 4 days. DHT concentrations in the incubation medium were chosen based on previous studies of tissue DHT levels in recurrent PCa [25].

### *2.4. Stimulation of Cell Growth by Treatment with Growth Factors and Cytokines*

Examination of the effects of growth factor and cytokine stimulation was performed as previously described [17], with minor modifications. E9 (5 × 10<sup>3</sup> cells/well), F10 (4 × 10<sup>3</sup> cells/well), and AIDL cells (6 × 10<sup>3</sup> cells/well) were cultured in phenol red (+) RPMI-1640 supplemented with 10% FBS for 2 days, and the culture medium was then replaced with phenol red (-) RPMI-1640 supplemented with 1% CS-FBS containing DHT (0.1 nM). One day later, the culture medium was replaced with phenol red (-) RPMI-1640 containing 10 ng/mL each of recombinant EGF, FGF-2, FGF-7, FGF-10, HGF, IGF-1, TGFβ1, VEGF, and IL-6. Cells were then incubated for 4 days before analysis.
