Supplement 2 – Sample Processing, Normalization, and Analysis
Enumeration of CTCs was done from 7.5 mL of blood drawn into a CellSave preservative tube
(Janssen Diagnostics). Blood samples were processed within 96 hours using the Cellsearch
System Epithelial Cell Kit (Janssen Diagnostics). In this system, epithelial cells are
immunomagnetically enriched from whole blood using anti-EpCAM antibodies loaded with
ferrofluid nanoparticles. Enriched cells are stained with the nuclear dye 4’,6-diamidino-2phenylindole (DAPI), anti-cytokeratin 8/18/19 labeled with phycoerythrin (PE), and anti-CD45
labeled with allophycocyanin (APC), followed by scanning using the CellTracks Analyzer
(Janssen Diagnostics). All cells ≥4 µm, with round-to-oval morphology, positive for cytokeratin
and DAPI, with at least 50% overlap in the DAPI and cytokeratin signal, and negative for CD45
were considered CTCs. All samples were analyzed by two independent, trained reviewers.
For the molecular characterization of CTCs, 7.5 mL of blood from an EDTA tube was processed
using the CellSearch Profile Kit (Janssen Diagnostics) within 24 h to limit mRNA degradation.
No staining step was performed after immunomagnetical enrichment. Instead, the buffer was
aspirated after incubation in a hand magnet and enriched cells were lysed in buffer RLT+
(Qiagen, Valencia, CA) and stored at -80°C until subsequent RNA isolation using the AllPrep
DNA/RNA Micro Kit (Qiagen). Of the resulting 12 µL with >200 bp RNA, 5 µL was used for
the generation of 10.5 µL cDNA (RevertAid H Minus First Strand cDNA Synthesis Kit from
Thermo Fisher Scientific, Waltham, MA), followed by an RNAse H step (Ambion, Life
Technologies) to degrade the remaining RNA. Next, 3 µL of the cDNA was used to specifically
pre-amplify the transcripts generated by the nine Taqman assays depicted in Supplementary
Table 1, which was done in 14 cycles according the protocol supplied by the manufacturer of the
Taqman PreAmp Master Mix kit (Life Technologies, Carlsbad, CA). Following pre-
amplification, the resulting 12 µL sample was 15-fold diluted prior to 35 cycles of RT-qPCR
using the Mx3000P Real-Time PCR System (Agilent, Amsterdam, The Netherlands). For each
sample, nine individual PCR reactions were performed in duplicate in a final volume of 20 µL
containing 5 µL diluted, pre-amplified cDNA, 30–50% (V/V) Taqman Universal Master Mix
(4326614, Life Technologies), and 0.5-1 µL Taqman gene expression assay. This was done in 35
cycles, according the protocol supplied by the manufacturer of the Taqman assays. Altogether,
1.5 μL of RNA from the original sample was used, which was further diluted for cDNA
synthesis and pre-amplification, leaving an average aliquot of ~11% of the original starting
material to measure the expression levels of AR-WT and AR-V7 using Taqman Gene Expression
Assays (Applied Biosystems, Carlsbad, CA; Supplementary Table 1). The amount of epithelial
cell input in the aliquot was calculated for each patient, using the average signal of EPCAM and
KRT19 from the aliquot, which correlated with the CTC count in 7.5 mL blood as assessed by
the CellSearch System from the parallel CellSave tube (Spearman's r=0.71; P<0.01;
Supplementary Figure 1A). The final epithelial cell input per patient can be found in
Supplementary Table 2. To correct for CTC count and epithelial tumor cell input, Cq values of
AR-V7 and AR-WT were normalized to the average Cq value of the epithelial genes EPCAM and
KRT19 and measured in the same PCR plate as follows: ΔCq AR = average Cq value of EPCAM
and KRT19 minus Cq value of AR-V7 or AR-WT. Three reference genes (GUSB, HMBS, and
HPRT1) served as the internal controls of isolated mRNA and cDNA quantity and quality.
Samples with an average reference gene Cq value >26.5, indicative for low and/or poor
RNA/cDNA quality, and/or an average epithelial gene Cq value >26.5, indicative for low/no
epithelial CTC input in the final RNA/cDNA sample, were excluded from the analyses.
Assay performance was tested through analysis of healthy blood donors (HBD) and cell line
experiments. All real time PCR assays were equally efficient both before and after preamplification (108%±4%, Supplementary Table 1). We measured the expression of AR-WT and
AR-V7 in pure cells of prostate (22RV1, LNCaP, PC3, and VCaP) and breast (CAMA1, MDAMB-415, MDA-MB-453, MPE600, SUM185PE, and ZR75.1) cancer cell lines. These were used
as negative and positive controls as follows: 22RV1 (WThigh/V7high), CAMA1 (WTlow/V7neg),
LNCaP (WThigh/V7low), MDA-MB-415 (WTlow/V7neg), MDA-MB-453 (WTlow/V7neg), MPE600
(WTlow/V7neg), PC3 (WTneg/V7neg), SUM185PE (WTlow/V7low), VCaP (WThigh/V7high), ZR75.1
(WTlow/V7low). Next, 100 cells of the same cell lines were spiked into 7.5 mL HBD blood,
followed by CellSearch enrichment, isolation of RNA, synthesis of cDNA, and PCR similar to
the patient samples and as described above. In Supplementary Table 2, the results from these
experiments are reported. To be able to assign patients as having AR-V7-positive or AR-V7negative CTCs, a cut-off value had to be established. As can be deduced from Supplementary
Table 2, the ΔCq value measured in the weakly positive breast cancer cell line ZR75.1 was 14.68. To maintain a certain margin, we decided to set the cut-off for positivity, i.e., any
detectable AR-V7 signal, at a ΔCq of -15.