Human bone metastasis-derived prostate cancer PC-3 cells (ATCC® CRL-1435) and non-tumorigenic human prostatic epithelial PWR-1E cells (CRL-11611) were obtained from ATCC (Manassas, VA, USA). PC-3 cells were routinely maintained in phenol red-positive F-12K modified medium (ATCC, USA) containing 10% FBS and 1% penicillin-streptomycin. PWR-1E cells were maintained in keratinocyte serum-free medium (Life Technologies, USA) supplemented with 50 μg/mL bovine pituitary extract, 5% L-glutamine, and 5 ng/mL epidermal growth factor. Cells were grown as monolayers in T-25 tissue culture flasks, in a humidified atmosphere containing 5% CO2 at 37℃ and passaged once/twice a week. For all the experiments, cells were harvested at low passage numbers: PC-3 cells between passages 28 and 31, and PWR-1E between passages 18 and 22.
Total RNAwas isolated from PC-3 and PWR-1E cells grown to approximately 80% confluence with TRIReagent (Ambion, Austin, TX, USA) following the manufacturer's instructions. Concentration and purity of total RNAwas assessed by spectrophotometry at 260/ 280 ratio with NanoDropTM (Thermo Scientific, USA). RNA samples from cell lines were processed for reverse transcription using the QuantiTect Reverse Transcriptase kit (Qiagen, Germantown, MD, USA). First, to eliminate any contaminating genomic DNA, 1 μg of total RNA was incubated with genomic DNA Wipeout Buffer (Qiagen, USA) for 2 minutes at 42℃. The reverse-transcription master mix, containing QuantiTect RT enzyme, QuantiTect RT Buffer and RT Primer Mix (oligo-dTand random primers), was prepared and added to the template RNA. Samples were incubated at 42℃ for 15 minutes, followed by inactivation at 95℃ for 3 minutes. A 20 μL final volume of cDNA was stored at - 20℃ until used in qPCR.
Transcript expression profiles for the currently described atypical chemokine receptor family were analyzed in the cell lines by quantitative PCR (qPCR) using QuantiTect SYBR Green PCR Master Mix (Qiagen, USA) and qPCR primer panels (OriGene Technologies, USA). Primers included in the qPCR primer panels are shown in Table 1. PCR for each sample was performed in triplicates with QuantiTect® SYBR® Green PCR Master Mix (Qiagen, USA) in a StepOne Real-Time PCR System (Applied Biosystems, USA), with an initial denaturing step at 95℃ for 15 minutes, followed by 40 cycles of amplification. Changes in gene expression for each target gene in metastatic PC-3 cell line were calculated with the Sequence Detection System 2.1 software (Applied Biosystems, USA) using the comparative CT method (2-ΔΔCT) and the reference non-tumorigenic cell line PWR-1E. Expression levels for each target gene were normalized to the expression levels of the reference genes HPRT1 and β-actin. Melting curves for all samples were acquired for quality control purposes. Statistical significance of differences in gene expression was calculated with the t-test using GraphPad Prism (GraphPad Software Inc, San Diego, CA, USA). A P < 0.05 was considered statistically significant.
Atypical chemokine receptors Primer pairs (Forward/Reverse) ACKR1 (DARC) 5'-GGGCTGAAGAAGGCATTGGGTA/CTTGGACCTCACCAGGAAATCC-3' ACKR2 (D6) 5'-GACTACGCACTCCAGGTAACAG/AAGCCTTCAGGTACTGGCGGAA-3' ACKR3 (CXCR7) 5'-CCAAGACCACAGGCTATGACAC/TGGTTGTGCTGCACGAGACTGA-3' ACKR4 (CCRL1) 5'-GTCTCTGGAATGCAGTTTCTGGC/GGTATGCTCAGCAAGATGGCAG-3' ACKR5 (CCRL2) 5'-TGCCGCTGTTTCCATCTGCGTA/ACACTTCGGTGGAATGGTCAGG-3'
Table 1. Primer sequences for PCR assays
Detection of CCRL2 was performed with antihuman-CCRL2 antibody (ab167114, abcam®) and EXPOSE Mouse and Rabbit Specific HRP/AEC Detection IHC system (ab93686, abcam®). Briefly, cells (1×104 cells/mL) were seeded on glass bottom Petri dishes (FluoroDish TM, World Precision Instruments, Inc, USA) in culture medium overnight. On reaching ~70% confluency, the dishes were rinsed twice with PBS and fixed in 4% paraformaldehyde/PBS for 10 minutes, and washed three times in PBS. Unspecific binding sites were blocked with protein block reagent for 10 minutes at room temperature. Primary mouse/ IgG, monoclonal, anti-human-CCRL2 antibody diluted in UltraClean Diluent (Thermo Scientific, USA) (1:100) was added and incubated at 4℃ overnight. Then, dishes were washed for 5 minutes three times with PBS-T, incubated 10 minutes with Mouse Specifying Reagent, rinsed 2 times in buffer, and HRP conjugate applied and incubated for 15 minutes at room temperature. After washing, antibody binding was detected with AEC chromogenic substrate (3-amino-9-ethyl-carbazole). Negative control slides were incubated in absence of primary antibody only. Nuclei were counterstained with hematoxylin (Sigma-Aldrich), dehydrated, and mounted. Intensity of staining was evaluated at 100× and 400× final magnification with an Eclipse 400 microscope connected to a DSFi1 camera (Nikon, Japan) by a pathologist who was blinded to the cell type on each slide. Staining intensity was scored using NISElements-3.0® software and classified as strong, moderate, weak, or absent.
CCRL2 protein expression was evaluated using a prostate tissue microarray (TMA) approach. For TMA construction, archived FFPE radical prostatectomy and trans-urethral prostatectomy tissue specimens were obtained from 47 patients with a previous diagnosis of localized prostate cancer, who underwent surgical resection as their primary treatment. For each patient, hematoxylin- and eosin-stained sections from donor blocks were subjected to pathological review to determine the presence of benign and cancerous tissue; then, matched tissue sections for benign prostate tissue (BPT) and cancerous prostate tissue (PCa) were cut out from the respective FFPE block for TMA. For each case, matched BPT sections were taken from regions distant to the PCa lesions. FFPE tissue specimens were obtained from the archives of the Department of Pathology at Hospital Universitario del Caribe with approval by local ethics committee and the studies were conducted according to the Declaration of Helsinki.
TMA design and construction were performed at the Sidney Kimmel Cancer Comprehensive Center, Johns Hopkins University (Baltimore, MD, USA). TMA construction was performed according to a previously described technique, using 0.6 μm cores of PCa and matched BPT from donor blocks. Four μm sections were cut off from the TMA block and stained with H&E to check the histopathology in each tissue core according to the previous design in the TMAJ® software. TMA processing was performed at Johns Hopkins University Oncology Tissue Services Core, Regional Oncology Research Center (Baltimore, MD, USA). The number of cores grouped by tissue type was 95 BPT and 136 PCa for a total of 231 cores.
To evaluate CCRL2 protein expression in PCa and BPT, 4 μm sections from the TMA block were subjected to immunohistochemistry. TMA sections were deparaffinized in xylene and rehydrated in alcohol according to standard procedures. Sections were steamed for 30 minutes in 10 μmol/L citrate buffer (pH 6.0) to unmask the epitopes, incubated in Hydrogen Peroxide Block and Protein Block (Abcam®, Cambridge, MA, USA), and then incubated with primary mouse/IgG, monoclonal, anti-human-CCRL2 antibody (ab167114, abcam®) diluted in UltraClean Diluent (Thermo Scientific, USA) (1:100) at 4℃ overnight. Then, slides were washed for 5 minutes three times with PBS-T, incubated 10 minutes with Mouse Specifying Reagent, rinsed 2 times in buffer, and HRP conjugate applied and incubated for 15 minutes at room temperature. After washing, antibody binding was detected with AEC chromogenic substrate (3-amino-9-ethyl-carbazole). Negative control slides were incubated in absence of primary antibody. All tissue slides were counterstained with hematoxylin, dehydrated, and mounted. Images from stained samples were captured at 100× and 400× final magnification using an Eclipse 400 microscope connected to a DS-Fi1 camera (Nikon, Japan). To quantify CCRL2 expression in the TMA, immunostaining was scored by microscopically assessing the percentage of luminal epithelial cells with positive staining to obtain a final H-Score. For this, 10 fields were chosen at random at 100× magnification and staining intensity in the benign luminal epithelial cells and tumor cells was scored as 0, 1, 2, or 3, corresponding to negative-, weak-, intermediate-, and strong-brown staining, respectively. The total number of cells in each field and the number of cells stained at each intensity score were counted. The average percentage positive was calculated and the following formula was applied: H-score = (% of cells stained at intensity category 1×1) + (% of cells stained at intensity category 2×2) + (% of cells stained at intensity category 3×3).
Differences in CCRL2 protein expression were assessed by comparing IHC scores (H-Scores) between BPT and PCa groups using Mann-Whitney test for nonGaussian distribution. Statistical analyses were performed using GraphPad Prism® v5.00 software (GraphPad Software Inc, San Diego, CA); P < 0.05 was considered statistically significant. Immunostaining intensity scoring in BPT and PCa regions was performed by two independent pathologists in blind.
RNA extraction and cDNA synthesis
Selection of specimens for inclusion into the TMA and ethical considerations
TMA design and construction
TMA immunohistochemistry and data analysis
Expression data for the atypical chemokine receptors analyzed by qPCR in PC-3 cells are presented in Table 2. Compared with PWR-1E cells, two out of five atypical chemokine receptor genes were overexpressed (ACKR3/CXCR7 and CCRL2) and the remaining three were underexpressed (ACKR1/ DARC, ACKR2/D6, and ACKR4/CCRL1) in PC-3 cells.
Atypical chemokine receptors PWR-1E CT PC-3 CT FC P-value ACKR1(DARC) 31.95 ND NA ACKR2(D6) 33.95 34.95 -4.70 0.0002 ACKR3 (CXCR7) 33.95 29.93 6.86 0.003 ACKR4(CCRL1) 30.95 32.94 -9.34 0.001 ACKR5 (CCRL2) 36.99 24.95 6758.56 0.0001 Note: Fold-change (FC) values were calculated using the 2–ΔΔCT method as described in Materials and methods. For fold changes < 1, the negative inverse of the result was reported as a fold decrease. P values were calculated by Student's t-test (two sided, for triplicate samples) from comparing fold changes in PC-3 cell lines relative to PWR-1E cell line. Expression levels for each target gene were normalized to the expression levels of the reference (housekeeping) genes HPRT1 and β-actin. Gene transcripts with CT (threshold cycles) > 37 were considered not detected (ND).
Table 2. Expression data analyzed by quantitative PCR in PC-3 cells
As mRNA for CCRL2 exhibited the most differential expression, it was chosen for further analysis at the protein level in the prostate cancer cell line PC-3 and the non-tumorigenic PWR1-E cells. Immunocytochemical staining revealed overexpression of CCRL2 in PC3 cells compared with the non-tumorigenic PWR-1E cells (Fig. 1). While non-tumorigenic PWR-1E cells did not exhibit cytoplasmic staining, PC3 cells had a strong cytoplasmic staining, indicating a high level of expression. The data indicated that CCRL2 staining was significantly stronger in the cancerous prostate cell line, compared to the non-tumorigenic cell line.
CCRL2 protein expression was evaluated by IHC in a TMA arrayed with 231cores from benign prostate tissue and prostate cancer tissue obtained from 47 patients. Immunohistochemical evaluation showed that CCRL2 staining was present predominantly in the cytoplasm of prostate cancer tumor cells. Compared with benign epithelial cells in BPT, CCRL2 expression was higher in epithelial tumor cells in cores with PCa (P < 0.0001). Mann-Whitney test showed a significant difference in staining between benign and cancerous tissues (P < 0.0001). There was a higher mean in immunohistochemical staining score in PCa (189.0, 95% CI: 176.6-201.4), compared with BPT (124.3, 95% CI: 115.0-133.6). The results of CCRL2 immunostaining in TMA are presented in Fig. 2.