Clone 80h3

CTCs were isolated from fresh blood specimens drawn from patients with prostate cancer, following negative depletion of leukocytes using the microfluidic CTC-iChip as reported previously^{26 (link),27 (link)}. Briefly, 10–20 ml of whole blood specimens were incubated with biotinylated antibody cocktails against CD45 (R&D Systems, clone 2D1), CD66b (AbD Serotec, clone 80H3), and CD16 (BD Biosciences), followed by incubation with Dynabeads MyOne Streptavidin T1 (Invitrogen) for magnetic labeling and depletion of leukocytes. After CTC-iChip processing, the CTC-enriched product was further stained with FITC-conjugated antibody against EpCAM (Cell Signaling Technology, clone VU1D9) and PE-conjugated antibody against CD45 (BD Biosciences, clone HI30). Single CTCs (FITC positive and PE negative) or white blood cells (WBCs, FITC negative and PE positive) were individually picked into PCR tubes containing 5 μl RNA/DNA lysis buffer using micromanipulator (Eppendorf TransferMan NK 2) and snap-frozen in liquid nitrogen. In total, 38 CTCs from 5 different patients (GU114, GU169, GU181, GU216 and GURa15) with metastatic prostate cancer were individually picked, sequenced and lineage-confirmed based on transcriptome and DNA copy number. One patient sample (GU169) had only one CTC, and it was therefore excluded from some downstream analyses focused on the four patients with multiple CTCs.