Radiolabeling of PSMA-HBED-CC
Elution of a 68Ge/68Ga-Generator (iThemba Labs, Cape Town, South Africa) was performed with 6 mL 1 M HCl, and the eluate was diluted with 6 mL H2O. 68Ga was retained on a cation exchange cartridge (Reagent and Hardware Kit SC-01, ABX, Radeberg, Germany) in a GRP Module (Scintomics, Lindach, Germany). M = 1200 (600–2000) MBq 68Ga was eluted from the resin with 1.5 mL 5 M NaCl. Radiolabeling of 5 μg (5.3 nmol) PSMA-HBED-CC in 3 mL 1.5 M HEPES (Kit components, ABX, Radeberg, Germany) was performed during 10 min at 95 °C in a reaction vessel for single use from the kit. Purification of the product was performed as already described by Eder et al. . Radiochemical purity was determined by TLC and HPLC (Additional file 1). In contrast to the described methods, we used an Agilent 1200 Series HPLC equipped with a 250 × 4.6 mm Lichrosorb RP C18, 5 μm column (NORDANTEC, Bremerhaven, Germany) with the described mobile phases (A aqueous 0.1 % trifluoroacetic acid (TFA), B acetonitrile with 0.1 % TFA) and a linear gradient starting with 100 % A to 90 % B after 12 min and followed by 3 min equilibration with 100 % A, flow rate 1 mL/min.
Cell culture subtypes
All cells were cultured in 25 cm2 culture flasks in 7-ml DMEM medium supplemented with 1 % l-glutamine (Lonza, Basel, Switzerland), 10 % FCS (PAA, GE Healthcare Bio-Sciences, Uppsala, Sweden) and 2 % sodium pyruvate (Gibco, Karlsruhe, Germany). Cell cultures were used below 40 passages after subtype development.
Castration-resistant prostate cancer (CRPC): the human cell line VCaP derived from a hormone-refractory bone metastasis was obtained from the American Type Culture Collection (ATCC; CRL-2876, Wesel, Germany). This cell line proliferates without androgen supply.
Revert castration-resistant prostate cancer (revCRPC): this cell line represents the pre-CRPC phenotype and was subcultured with 1 nmol/L testosterone (Sigma, St. Louis, MO, USA) over at least 20 passages resulting in a steady androgen-sensitive subtype for experiments within the subsequent 10 passages .
Abiraterone acetate tolerant castration-resistant prostate cancer (CRPCAA): CRPCAA was obtained by sub-culturing CRPC with 5 μmol/L abiraterone acetate over at least 20 passages.
Western blot analysis of the cell culture subtypes
Total protein lysates were prepared using RIPA buffer with protease inhibitors (Roche, Mannheim, Germany) and were quantified using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, California, USA). The samples were prepared for gel electrophoresis by mixing each with 4× Laemmli buffer (Bio-Rad). Each well of the gel was loaded with 15 μg protein. The gel was run for approximately 30 min at 250 V in the Mini-PROTEAN® Tetra System with the POWER PAC 300 (both Bio-Rad). The blotting step was carried out with the Bio-Rad Trans-Blot®Turbo™ RTA Transfer kit onto a nitrocellulose membrane. The membrane was blocked with 5 % milk powder in TBS buffer with 0.5 % Tween for 1 h at room temperature. For protein analysis, the primary antibody against PSMA (Dako M3620, monoclonal mouse, diluted 1/500, overnight at 4 °C) was used. The housekeeping gene α-tubulin (monoclonal mouse, diluted 1/5000) was labelled at room temperature for 1 h. The primary antibodies were detected with HRP-labelled anti-mouse secondary antibodies (Dako, dilution 1/1000). The signal development was made visible by applying a chemiluminescent substrate (ECL system by Amersham Bioscience, Freiburg, Germany).
Immunohistochemical analyses were performed as described previously . Adherent tumour cell lines were trypsinised from culture flasks and washed with PBS. Cells were spun down at 300g for 5 min. The pellet was fixated with 1 ml 4.5 % formalin for 1 min through resuspension. After another centrifugation, the formalin was removed, and the cell pellet was resuspended in 200 μl liquid 3 % agarose gel. The gel pellets were dried overnight at room temperature, then the hardened gel cones were removed from the cups and cut lengthwise. These gels cones were put into tissue capsules for the pre-treatment and following paraffin embedding. Paraffin blocks were cut at 3 μm for immunohistochemistry. Sections were stained using a Dako autostainer with the Dako EnVision™ FLEX+ detection system (Dako, Glostrup, Denmark). The system detects primary mouse and rabbit antibodies, and the reaction was visualised by EnVision™ FLEX DAB+ Chromogen. Using EnVision™ FLEX+ Mouse (LINKER) or EnVision™ FLEX+ Rabbit (LINKER) (Code K8019), signal amplification of primary antibodies can be achieved. Deparaffinisation, rehydration and heat-induced epitope-retrieval (HIER) was carried out in one step with the 3-in-1 procedure buffer (Dako, Glostrup, Denmark, Target Retrieval Solution), pH 9 high ((10×) (3-in-1) Code S2375)) at 97 °C using a PT Link, Pre-Treatment Module 6 (Dako). Tissue samples were analysed by light microscopy after 8 min counterstaining with Meyer’s haematoxylin (Dako). As primary antibodies, we used anti-PSMA (Dako M3620, monoclonal mouse antibody, diluted 1:50, 30 min).
Uptake measurements and cell count
All uptake experiments were carried out in three separate series resulting in n = 9 culture flasks for each cell line, variation in medication and measurement time. To assess the capacity of cells for the labelled compounds in one series, 0.3 nmol/L 68Ga-labelled peptide (100-300 kBq/ml) was simultaneously applied to three parallel cells cultures for each interval (1–3 h) and each cell line. Since activity yield from generator, yield of synthesis as well as delay between synthesis and cell culture experiments differed between the experimental series, peptide amount kept constant between the separate series. Before measuring the tracer uptake, culture medium was discarded, and the cells were washed twice with phosphate buffered saline (PBS) buffer pH 7.4. The cells were dissociated by accutase/EDTA (PAA, GE Healthcare Bio-Sciences, Uppsala, Sweden) and centrifuged. Activity of cell pellet and collected supernatants was measured. Data were half-life-corrected, and percentage uptake of applied activity was calculated. Since differences in treatment resulted in altered proliferation, the calculated uptake was correlated to the determined cell count in each particular culture flask and normalised to 106 cells as already described [26, 27].
Variations of abiraterone acetate and testosterone treatment
CRPC and revCRPC cells were incubated over 48 h with 5 μmol/L Abiraterone acetate (Janssen-Cilag, Neuss Germany) prior to activity supply. In parallel revCRPC cultures abiraterone acetate (AA)-induced androgen deprivation was aggravated by testosterone withdrawal. In CRPCAA cells, AA was eliminated from culture medium and 1 nmol/L testosterone was supplied.
Analysis of mRNA expression
mRNA expression of 106 cells per sample were analysed by qRT-PCR. Total cellular RNA from cultured cells was extracted with the Quick-RNA™MiniPrep (Zymo Research, Freiburg, Germany). Total RNA integrity and quantity were assessed on an Experion™ Automated Electrophoresis System with analysis kits consisting of microfluidic chips and reagents (Bio-Rad, Munich, Germany). Reverse transcription of 500 ng total cellular (tc) RNA was performed with random hexamer primers and an Omniscript RT Kit (QIAGEN, Hilden, Germany). Expression analyses were processed on a CFX96™ real-time detection system (Bio-Rad, Munich, Germany) with SsoAdvanced Universal SYBR Green supermix. The 20 μl reaction mix from the kit was supplemented with 2 μl cDNA, 0.6 μM gene-specific primers (IBA, Goettingen, Germany). Primers were designed by Primer 3 [http://primer3.ut.ee] and assessed for PCR efficiency.
Time courses of gene expression (Fig. 3) were calculated as percent expression using the analysis of relative gene expression by real-time quantitative PCR and the 2(-ΔΔC(T)) method . The housekeeping gene for calculation of relative expression was acidic ribosomal protein (ARP) and for proof of principle of this calculation another housekeeping gene ribosomal protein L13a (RPL) was used.
To elucidate if the three different subtypes or modification of medication resulted in different internalisation rates, a protocol for the detection of endocytosis in adherent cell lines was modified . CRPC, CRPCAA, rev-CRPC as well as rev-CRPC under ADT (with abiraterone acetate and testosterone withdrawal) were investigated. Therefore, three cultures flasks (25 cm2) for each subtype and modification were seeded with 3 × 105 cells and allowed to attach overnight in their specific mediums as described above. All cultures were grown for an additional 48 h whereby ADT was performed for three flasks as already described above. Cultures were incubated with 0.3 nmol/L 68Ga-HBED-CC for 5 h. In contrast to the mentioned protocol, adherent cells were harvested with trypsin/EDTA (PAA). Total activity was determined in culture medium, in the fraction of stripped radiolabel from cell surface proteins and the harvested cells. Thereafter, cells were spun down and total cell count was determined. Results were calculated as percentage of fraction activity normalised to 106 cells of applied activity after half-life correction.
Statistical analyses (Statistical Analysis Software: SPSS Statistics 22.0 for Windows) were performed using non-parametric tests (Wilcoxon, Mann-Whitney). Means (m) and standard deviations (SD) were calculated. Differences were considered significant when p < 0.05.