Sc 17750

Informed consent was obtained to collect human mCRPC tissues and generate the patient-derived xenograft tumors as described previously (male CB17 SCID mice between 4–6 weeks of age; maximum tumor size: 1000 mm³; housing ambient temperature: 20–26 °C; ambient humidity: 30–70%)^{37 (link),38 (link)}. The study was approved by the University of Washington Human Subjects Division institutional review board (no. 2341). All animal studies were approved by University of Washington IACUC and performed according to NIH guidelines. Molecular characterization of AR⁺ mCRPC LuCaP PDXs 70CR, 78CR, 81CR, 96CR, 105CR, 136CR and 147CR was previously described^{37 (link),38 (link)}. LuCaP PDX 167CR was established from a liver metastasis of a male who died of abiraterone-, carboplatin- and docetaxel-resistant CRPC. LuCaP 167CR expresses AR (mouse monoclonal [F39.4.1] anti-AR; #MU256-UC, Biogenex; dilution 1:60), responds to castration and is negative for synaptophysin (mouse monoclonal [D-4] anti-synaptophysin; #sc-17750, Santa Cruz Biotechnology; dilution 1:200). PDX cellular morphology recapitulates the original liver metastasis (Supplementary Fig. 8a; characterization as previously described^{37 (link)}).

IHC was performed on paraffin-embedded tissue sections. After deparaffinization, rehydration, and antigen retrieval with citrate buffer (Invitrogen), slides were permeabilized with 0.5% triton X-100. Slides were then blocked using a ready-to-use IHC kit (BioVision) as described by the manufacturer. Slides were then incubated with primary antibodies: anti-CXCR7 (1:50; R&D, MAB42273), mouse IgG control (1:100; R&D, MAB002), anti-phospho-Aurora A (Thr288) (1:1000; CST, 3079), mouse anti-SYP (1:500; Santa Cruz, sc-17750), and anti-AR (1:200; Santa Cruz, SC-186) overnight at 4°C in a humidity chamber. Slides were then washed with 1 × TBS 3 times for 5 minutes each time. For secondary antibodies, slides were incubated with IBSC-1-step HRP-anti-mouse, rat, and rabbit polymer provided in the kit. Slides were washed again with TBS (3 times for 5 minutes each time), then incubated with 3,3′- Diaminobenzidine (DAB) substrate for 1–5 minutes at room temperature. Slides were then counterstained with hematoxylin for 15 seconds, washed with running tap water, dehydrated in ethanol, cleared with xylene, and mounted with Permount (Fisher Chemical). Slides were visualized and imaged with an Olympus BX41 microscope bound to with Olympus UTV 0.5XC3 camera.