22RV1, LNCaP, VCaP, PC3 and DU145 prostate cancer cell lines are here referred to as 22RV1AR+/AR-V7+++, LNCaPAR+/AR-V7-, VCaPAR+++/AR-V7+, PC3AR(+)/AR-V7-, DU145AR-/AR‑V7- according to their published and in this study validated AR-FL and AR-V7 expression [18-20] and cells were grown in DMEM supplemented with 10% Foetal Bovine Serum (FBS), 2mM L-Glutamine, 4nM HEPES or MEM supplemented with 10% FBS, 2mM L-Glutamine, 4nM HEPES at 370C in 5% CO2. Cell lines were obtained directly or through Australian distributers (Merck/Sigma-Aldrich, Castle Hill; Invitro, Lane Cove, Australia) of the European Collection of Authenticated Cell Cultures (ECACC) or American Type Culture Collection (ATCC) and tested to be mycoplasma free (MycoAlert Mycoplasma Detection Kit, Lonza, Rockland, USA) and STR authenticated (AGRF, Melbourne, Australia). Cells were seeded at approximately 30-40% confluency and harvested after 72-hour culture for immunoblotting and gene expression analysis.
Six rabbit anti-human-AR-V7 antibodies were compared in this study: clone EPR15656 (Abcam, VIC, Australia), clone E308L and “polyclonal antibody” (Cell Signalling, Danvers, MA, USA), clone SN8 (Creative Diagnostic, Shirley, NY, USA), clone DHH-1 (RQ4683, Assay Matrix, VIC, Australia), and clone RM7 (RevMab Biosciences, San Francisco, CA, USA), as well as the mouse anti-human-AR-V7 clone AG10008 (Precision Antibody, Columbia, MD, USA). The available information of antigens used for the anti-AR-V7 antibody generation is shown in Figure 1A. Additional antibodies used in this study are: mouse anti-human AR-FL, clone ER179 (Abcam, NSW, Australia), rabbit anti-GAPDH clone 14C10 (Cell Signaling, VIC, Australia), Alexa fluor 488 goat anti-rabbit IgG (H+L) (LOT 1423009) or Alexa fluor 488 goat anti-Mouse (H+L) (LOT 1252783) (Life technologies, Eugene, OR, USA), horseradish peroxidase-labelled donkey anti-Rabbit IgG (1:1000 dilution) (Lot 9526417, GE Healthcare, Buckinghamshire, UK) or sheep anti-mouse IgG, Horseradish Peroxidase linked F(ab’)2 fragment (1:1000 dilution) (Lot 312511, Amersham, GE Healthcare, Buckinghamshire, UK) and Alexa fluor 555 Phalloidin (Abcam, NSW, Australia).
Droplet digital PCR (ddPCR)
In brief, total RNA was extracted with ISOLATE II RNA Mini Kit (Bioline, London, UK) from approximately 5x106 cells. Quality and quantity of RNA was tested using a fragment analyser (5200 Fragment Analyzer System, CA, USA). cDNA was synthesised from 1µg of total RNA per cell line using the SensiFAST cDNA Synthesis Kit (Bioline, London, UK). ddPCR to detect AR-V7 and full-length AR (AR-FL) was performed as described previously . Quality of RNA was confirmed by conducting GAPDH ddPCR as described previously .
Approximately 1x106 cultured cells were harvested and lysed in RIPA lysis buffer (50mM Tris-Cl pH7.5, 150mM NaCl, 0.5% Triton X-100, 2mM EDTA, 2mM EGTA 25mM NaF, 10% glycerol) containing 1x protease inhibiters (Roche, Basel, Switzerland) for 30 minutes placed on ice, followed by maximum microfuge centrifugation speed (11,700g, 4oC, 20) minutes and recovery of supernatant. Protein concentrations were determined using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). 30µg total protein per sample was separated on 4-12% Bis-Tris gels (Invitrogen, Life Technologies) and transferred to Polyvinyl difluoride (PVDF) membrane (Amersham, GE Healthcare, Buckinghamshire, UK). Membranes were incubated with primary antibodies (dilutions see SuppTable 1) overnight under gentle agitation at 4oC. After three Tris-buffered saline with 0.1% Tween 20 detergent (TBS-T) washes, membranes were incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:1000 dilution) or sheep anti-mouse IgG, horseradish peroxidase linked F(ab’)2 fragment (1:1000 dilution) for 1 hr at room temperature and again washed three times. Membranes were developed using Western Lightning TM Plus-ECL Enhanced Luminol Reagent Plus (LOT 275-13481) and Western Lightning TM Plus-ECL Oxidizing Reagent Plus (LOT 265-13481) (PerkinElmer, Waltham, MA, USA) and imaging was performed with an Odyssey imager (LI-Cor Biosciences, Lincoln, NE).
Immunocytostaining of Cell Lines
For each cell line approximately 20,000 cells were seeded on sterile, round coverslips in 12-well plates and cultured for 72 hours followed by fixation with 3.7% paraformaldehyde for 10 minutes. Cells were permeabilized with 0.2% Triton-X for 10 minutes and blocked using 10% goat serum in PBS for 30 minutes. Primary antibodies were diluted in 0.5% FBS in PBS (Supplementary Table 1) and incubated for 1 hour. Secondary antibodies conjugated with Alexa fluor 488 goat anti-rabbit IgG (H+L) (1:5000) or Alexa fluor 488 goat anti-mouse (H+L) (1:5000) were diluted in PBS with 0.5% goat serum and incubated 30 minutes. Cells were stained with Alexa fluor 555 phalloidin for 0.5 hours followed by nuclear staining by using 1x Hoechst (Fluxion, San Francisco, CA, USA) in PBS for 10 minutes. Coverslips were mounted with Pro Long TM Glass Antifade Mountant (Eugene, OR, USA). Images were taken with Olympus IX71 microscope (Olympus, Tokyo, Japan) at 20x magnification with consistent exposure times.
CTC Enrichment and Immuncytostaining
For each patient, 2x 9 mL peripheral blood was collected into 2 EDTA vacutubes (Greiner Bio-One) and processed within 24 hours. 2x 9mL blood was used to isolate CTCs using RosetteSepTM CTC enrichment cocktail containing anti-CD36 (Stemcell Technologies, Victoria, Australia) according to supplier’s instructions. In brief, blood was incubated with antibody cocktail for 10 minutes and then diluted with 2% FBS in PBS as recommended by manufacturer, transferred to a Sepmate tube containing lymphoprep density gradient medium (Stemcell technologies, VIC, Australia) and centrifuged at 1200xg for 10 minutes. The supernatant with cellular layer was recovered and topped up to 50mL with 2% FBS in PBS and gently mixed. After a 10-minute 300xg spin, the supernatant was discarded, and cells were suspended in residual fluid by gentle tapping. Cells were washed once with PBS and spun again (300xg, 10 minutes), resuspended in 1.5mL PBS and transferred to a well of a 24-well glass bottom plate (Greiner Bio-One GmbH, Frickenhausen, Germany) coated with 3.5ug of CellTak (FAL354240, InVitro technologies, VIC, Australia) per cm2. After spinning the cells onto the glass (200x g, 10min) immunocytostaining was essentially performed as above including probing for CD45 to exclude lymphocytes and Hoechst to secure nucleated cellular identity.
Image analysis and Statistics
Image J (1.53c, National Institute of Health, USA) was used for RGB stacking and merging of images before doing quantitative image analysis using CellProfiler (Broad Institute, MIT, Massachusetts, USA) an automated image analysis software to measure biological phenotypes in images . CellProfiler segmented cell data for at least 150 cells per sample (nucleus and cytoplasm) based on staining and extracted data on nucleus, cell body and cytoplasm and AR-V7 intensity were saved in excel to transfer to Konstanz Information Miner (Knime) . The quantitative data from CellProfiler was used in Knime to compare the intensity of AR-V7 detected by different antibodies as well as cellular localization of AR-V7.