The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis

Antibodies targeting CD164 were generated in the laboratory of Dr. H J Bühring (Tübingen, Germany) and were described in detail previously [23]. These include antibodies targeting the class I CD164 epitope (clone 105A5, mIgM), the class II epitope of CD164 (clone 103B2, mIgG3) and the CD164 class III epitopes (Clones 67D2, mIgG1 and N6B6, IgG2a). In some experiments antibody from the N6B6 clone was purchased from BD PharMingen (San Jose, CA, USA) as well as murine isotype matched monoclonal controls. The control antibodies for these investigations included IgM clone A57H (Dako, Denmark) IgG3 clone 133316 (BD Pharmigen), IgG1 clone 11711, IgG2a clone 20102 (R&D Systems).

Prostate cancer cell lines (PC3, LNCaP C4-2B, and LNCaP), and bone marrow endothelial cells (HBME) were cultured in RPMI medium 1640, supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% l-glutamine (Invitrogen, Carlsbad CA). PC3 cells were originally isolated from a vertebral metastasis and were obtained from American Type Culture Collection (Rockville, MD). LNCaP cells were originally isolated from a lymph node of a patient with disseminated bony and lymph node involvement. The LNCaP C4-2B cells were derived from the parental LNCaP cell line by serial passage in mice to obtain a more metastatic cell line [30]. The HBME cells were isolated from a Caucasian male and immortalized with SV40 large T-antigen [23]. All cultures were maintained at 37°C, 5% C0₂, and 100% humidity.

Secondary data analysis was performed on data published by Dhanasekaran et al. [19] as previously reported [8]. Using a 10 K human cDNA micoarray which included ~5,520 known, named genes and 4,464 ESTs, the authors determined the gene-expression profiles of RNA obtained from more than 50 normal and neoplastic prostate specimens [19]. Supplementary information from Dhanasekaran et. al. were imported into a Microsoft Excel database and data clustered into normal associated tissues (NAP), benign hyperplasia (BPH), localized cancer (PCa) and metastasis (Met) [19]. The normalized mean and standard deviation values for CD164 was evaluated for significance with Instat 4.0 (GraphPAD software) using one-way analysis of variance (ANOVA), with the level of significance set at p < 0.05.

To determine which of these genes are responsive to CXCL12 and represent known adhesion molecules, RNA from three independent studies, carried out in triplicate and pooled, was analyzed from CXCL12 treated LNCaP and LNCaP C4-2B cells (200 ng/ml, 2 hours,). The RNA was compared in triplicate against non-treated cells by DNA micro array using the Human genome U133 gene chips (Affymetrix Corp. Santa Clara, CA). Protocols and instrumentation setups, including total RNA samples, hybridization to the human micro arrays, washing, staining, and scanning were performed as recommended (Gene Chip Micro arrays; Affymetrix, Santa Clara, CA) by the University of Michigan Dental School Microarray Facility. The resulting arrays were scanned (HP Gene Array Scanner; Hewlett Packard, Palo Alto, CA), and data analysis performed first with the accompanying software to obtain average difference intensities (Gene Chip Expression Analysis Software, ver. 3.3; Affymetrix). Later, average gene intensity values were determined using a software package (DNA-Chip (dChip version 1.1)) [26] in consultation with the University of UMCCC Microarray Core Facility.

The prostate cancer cell lines at confluence were transferred to serum free media, and stimulated with CXCL12 at 0 or 200 ng/ml for 0.5–8 h. RNA was recovered using Trizol^R according to the directions of the manufacturer (Invitrogen, Carlsbad, CA). RNA integrity and purity was evaluated by electrophoresis with ethidium bromide and absorbance at A₂₆₀/A₂₈₀. 1.0 μg RNA, 10X RT buffer (1X RT buffer: 50 mM Tris, pH 8.3, 50 mM KCl, 8.0 mM MgCl₂, and 10 mM dithiothreitol), 25 mM dXTP mix (25 mM of each dXTP (ACGT)), 3.0 μg oligo d(T), and 2.5 U Reverse Transcriptase (M-MLV Reverse Transcriptase, Invitrogen, Carlsbad, CA) were incubated at 38°C for one hour. Amplification primers were designed using PrimerExpress™ software (Applied Biosystems, Foster City, CA) to cross intron/exon boundaries. Cross-reactivity was determined using BLAST [27], those with the lowest potential were synthesized. One-fifth of the double stranded product was then mixed with 10X Taq/RT buffer (1X Taq/RT buffer: of 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, and 2.0 mM dithiothreitol), 1 mM dXTP mix, 500 ng of each sense and antisense oligonucleotide, and 2.5 U Taq polymerase (AmpliTaq DNA Polymerase; Perkin Elmer Cetus, Norwalk, CT). Five pairs of forward and reverse primers were designed to identify the alternatively spliced transcripts of the CD164 gene and are presented in Table 1[20]. The samples underwent thermal cycling at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 minutes for 35 cycles, followed by a 10-min extension at 72°C (Perkin Elmer Cetus DNA thermal cycler). The PCR product of PC3, LNCaP C4-2B, and LNCaP cell lines were analyzed on 12% polyacrylamide gels and ethidium bromide stained.

Real time-PCR was also performed using random hexamers and 15.0 μl of SYBR^® Green PCR Master for quantitative, two step real-time RT-PCR (Applied Biosystems, Foster City, CA) with 100 nM of the E1 and E2 primers (Table 2) and 2 μl of the RT product in a total volume of 30 μl. The 2^nd step PCR reaction (95°C for 30 seconds, 60°C) was run for 40 cycles after an initial single cycle of 95°C for 15 minutes to activate the Taq polymerase. The PCR product was detected as an increase in fluorescence using an ABI PRISM 7700 instrument (Applied Biosystems, Foster City, CA). The mRNA levels were expressed as relative copies (% control) normalized against 18S mRNA (Ambion QuantumRNA™ 18S Universal Primers) and a standard curve constructed from serial dilutions of a purified CD164 cDNA fragment cloned by classical PCR.

High-density tissue micro arrays were constructed from clinical samples obtained from a cohort of over 600 patients, who underwent radical retro pubic prostatectomy at the University of Michigan as a primary therapy (i.e., no preceding hormonal or radiation therapy) for prostate cancer and from materials obtained from the University of Michigan Rapid Autopsy Program. The arrays were provided from the University of Michigan Comprehensive Cancer Center Tissue Core of the Prostate Specialized Program of Research Excellence program as detail previously [28]. Tumors were graded using the Gleason grading system and examined to identify areas of benign prostate, prostate cancer and bone metastasis. The formalin-fixed, paraffin-embedded tissues were deparaffinized and placed in a pressure cooker containing 0.01 M buffered sodium citrate solution (pH 6.0), boiled and chilled to room temp for antigen retrieval. The slides were incubated overnight at room temperature with anti-human CD164 antibody (BD Biosciences, San Jose, CA) diluted 1:100 (10 mg/ml), IgG2a (Clone 20102, R&D Systems, Minneapolis, MN). For PSA levels, antigen retrieval was performed in citrate buffer at pH 6.0. For AR staining, antigen retrieval was performed with EDTA at pH 8.0 in a pressure cooker. Polyclonal antibodies to PSA (1:2000 dilution, Dako Cytomation, Carpenteria, CA) and a monoclonal AR-clone AR 441 (1:50 dilution, NeoMarkers, Fremont, CA) were used for staining as previously reported [29]. A streptavidin/biotin detection method with 3,3'-Diaminobenzidine Tetrahydrochloride (DAB) was employed for signal detection and Harris hematoxylin was used as a counter-stain. Digital images were then acquired with the BLISS Imaging System (Bacus Laboratory, Lombard, IL). Immunostaining intensity was scored by a genito-urinary pathologist: as absent (1), weak (2), moderate (3) or strong (4). Scoring was performed in a blinded fashion using the Profile web based telepathology system without knowledge of overall the tumor grade, tumor size or clinical outcome [30, 31].

Prostate cancer cell lines were labeled with Vybrant CFDA SE Cell Tracer Kit, (Molecular Probes, Inc., Eugene, Oregon) for 30 min in RPMI according to the recommendations of the manufacturer and washed, and rested for 30 min. CXCL12 pretreatment was performed by incubating the CaP cells with PBS or 200 ng/ml CXCL12 (or 200 ng/ml CXCL12 that had been boiled for 15 min as a negative control), for 30 min at 37°C. Anti-CD164 mAb (N6B6) or an IgG2a isotype matched control mAb (R&D Systems) were added at 0 and 50 μg/ml to block adhesion. The prostate cancer cells (1 × 10⁵) were then directly deposited onto confluent HBME monolayers and cell-to-cell adhesion was preformed for 30 min at 37°C. Adherence was quantified in a 96-well fluorescent plate reader (IDEXX Research Products, Westbrook, ME), and compared to input fluorescent levels. Data are presented as percent change +/- standard deviation compared to non-treated controls.

Cell invasion into a reconstituted extracellular matrices costing of Matrigel™ overlaid on 8 μM pore sized in polyethylene terephthalate membranes was performed in dual chambered invasion plates (BD Biosciences, San Jose, CA). Test cells were placed in the upper chamber (1 × 10⁵cells/well) in serum free RPMI containing 0.1% BSA, and PBS or 200 ng/ml CXCL12 was added to the top or bottom chambers. Spontaneous invasion was compared to invasion supported by a CXCL12 gradient [32]. Anti-CD164 mAb (N6B6) (BD PharMingen, San Jose, CA, USA) or an IgG (R&D Systems. Minneapolis, MN, USA) control was added at 50 μg/ml to both chambers to block invasion. At the termination of the assay (24 h), the chambers were removed and 40 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml, Sigma, St. Louis, MS) was added to the top well and 80 μl of MTT added to the bottom wells, and incubated for a further 4 h at 37°C. After completely removing residual medium or cells from the top chamber, the purple residues attached to the bottom of the invasion chambers and those residues in the invasion matrix and adherent to the bottom of the upper chamber were released with 1 ml isopropanol (Sigma). The invasion chambers were rocked for 30 min at a medium speed, and 100 μl from each well read on a multi-well scanning spectrophotometer (Molecular Devices Corp. Sunnyvale, CA.) at OD₄₅₀.

PC3 and LNCaP C4-2B cells were cultured in Lab-Tek II 4-chamber slides (Nalge Nunc International, Naperville, IL, USA) at 5 × 10⁴ cells/chamber. After 24 h the cells were incubated with PBS or CXCL12 (200 ng/ml) for 2 h, then rinsed three times with ice cold-PBS, fixed in 4% paraformaldehyde for 25 min at room temperature, washed and endogenous peroxidase activity quenched with 75 mM NH4Cl and 20 mM Glycine in PBS at room temperature for 10 minutes. Thereafter, the cells were rinsed with PBS three times. Primary antibody incubations at a 1:50 dilution in PBS using the N6B6 clone or an IgG2a matched isotype control (clone 20102, R&D Systems) for 1 h at room temperature. Antibody detection was performed using an HRP-AEC staining kit using anti-mouse biotinylated antibodies (R&D Systems), and counter stained with hematoxylin (Sigma).