SUPPLEMENTARY TABLES

Supplementary Table 1. Assay development for the AR marker using the CellSearch System. Cells were spiked into 7.5mL of healthy volunteer blood and processed with the CellSearch Circulating Tumour Cell Kit. A user-defined assay was performed by adding the AR-AF488 antibody at a fixed concentration of 0.5µg/mL (diluted in 1x PBS) and a permeabilisation buffer containing saponin (Perm/Wash™ Buffer) at increasing concentrations (0x, 5x and 10x). We detected most AR-positive LNCaP cells when Perm/Wash Buffer was added at a concentration of 5x (24% AR-positive, AR Median intensity (MI)= 18), therefore confirming that cells need to be maintained in a permeabilised state during antibody incubation in order to achieve the best intracellular/nuclear staining.

Sample Nr. / Cell Line / AR-AF488 (µg/mL) / Concentration of Perm/Wash
Buffer / AR Positive (%) / AR MI
(Nuclear)
1 / LNCaP / 0.5 / - / 9% / 9
2 / LNCaP / 0.5 / 5x / 24% / 18
3 / LNCaP / 0.5 / 10x / 1% / 9
4 / PC3 / 0.5 / - / 21% / 13
5 / PC3 / 0.5 / 5x / 0% / 5
6 / PC3 / 0.5 / 10x / 7% / 8

Abreviations: AR-AF488 – Androgen Receptor conjugated to AlexaFluor 488; MI – Median intensity

Supplementary Table 2. Establishment of an optimal AR antibody concentration using the CellSearch System. We determined that the optimal concentration of AR-AF488 antibody (diluted in 1x PBS and 5x Perm/Wash Buffer) in blood samples spiked with LNCaP cells incubated with a series of concentrations ranging from 0.5µg/mL to 2.5µg/mL. The highest percentage of positive cells and AR median intensity was obtained when the AR-AF488 antibody was used at 2.5µg/mL (69% AR-positive, AR MI= 42).

Sample Nr. / Cell Line / AR-AF488 (µg/mL) / Concentration of Perm/Wash
Buffer / AR Positive (%) / AR MI
(Nuclear)
1 / LNCaP / 0.5 / 5x / 24% / 18
2 / LNCaP / 1.25 / 5x / 46% / 23
3 / LNCaP / 2.5 / 5x / 69% / 42
4 / PC3 / 0.5 / 5x / 0% / 5
5 / PC3 / 1.25 / 5x / 2% / 6
6 / PC3 / 2.5 / 5x / 3% / 9

Abreviations: AR-AF488 – Androgen Receptor conjugated to AlexaFluor 488; MI – Median intensity

Supplementary Table 3. Comparison of the number of CTCs in 7.5mL blood between the standard CellSearch protocol (no marker) and the CellSearch AR assay in two contemporaneously collected samples from 14 patients. Differences between CTC count values between the standard CellSearch protocol and the CellSearch AR assay were not significantly different (P=0.5085, Wilcoxon’s signed-ranked test). Only in 1 of the 14 samples (Patient 7) the classification changed from favourable (4 CTCs) to unfavourable (8 CTCs) when the AR antibody was added.

Patient ID / Standard CellSearch Protocol (no marker) / CellSearch AR assay
1 / 5 / 6
2 / 17 / 11
3 / 4 / 3
4 / 32 / 28
5 / 64 / 61
6 / 41 / 30
7 / 4 / 8
8 / 41 / 37
9 / 0 / 1
10 / 13 / 14
11 / 47 / 44
12 / 40 / 53
13 / 44 / 29
14 / 19 / 53

Supplementary Table 4: Intra-day* and inter-day+ assay variability

Patient 1* / Patient 2+ / Patient 3+
Run 1 / Run2 / Day 1 / Day 3 / Day 1 / Day 3
Number aCTC / 110 / 123 / 47 / 48 / 185 / 184
AR Mean Intensity / Minimum / 0.09979 / 1.084 / 1.425 / -0.9167 / -1.357 / -1.401
25% Percentile / 12.31 / 10.17 / 7.774 / 2.449 / 11.47 / 4.817
Median / 26.01 / 24.03 / 26.33 / 11.88 / 26.11 / 21.28
75% Percentile / 45.78 / 57.04 / 55.99 / 53.41 / 65.56 / 62.77
Maximum / 471.7 / 255.7 / 211.9 / 209.3 / 334.2 / 288.6

Abreviation: aCTC – unbiased automated circulating tumour cell count

Supplementary Table 5: Intra-run variability

Patient 2 / Patient 3
Sample 1 / Sample 2 / Sample 1 / Sample 2
Number aCTC / 47 / 35 / 185 / 170
AR Mean Intensity / Minimum / 1.425 / 0.4034 / -1.357 / 0.3122
25% Percentile / 7.774 / 5.653 / 11.47 / 10.29
Median / 26.33 / 18.50 / 26.11 / 25.25
75% Percentile / 55.99 / 61.18 / 65.56 / 62.62
Maximum / 211.9 / 195.2 / 334.2 / 301.5

Abreviation: aCTC – unbiased automated circulating tumour cell count