Human HPC KG1a cells and murine monocytic WEHI-3 cells (both from ATCC®, Manassas, VA) were maintained in RPMI-1640 with glutamine/10% FBS/1% penicillin/streptomycin (P/S) (all from Gibco™ Invitrogen Corp.; Grand Island, NY). Human prostate tumor MDA PCa 2b cells derived from bone metastases (26
) were propagated in BRFF-HPCI (AthenaES™, Baltimore, MD)/20% FBS/1% P/S. Other human bone-metastatic prostate tumor cell lines, PC-3, PC-3M, PC-3M Pro-4 and PC-3M LN-4 (27
), were maintained in RPMI-1640 with glutamine/10% FBS/1% P/S, while PC-R1 and PC-E1 cell lines (generously provided by Dr. Klaus Pantel; Hamburg, Germany) (29
) were cultured in RPMI-1640 with glutamine/10% FBS, 1% P/S, 10μg/ml transferrin, 5μg/ml insulin, 10ng/ml recombinant human EGF and 10μg/ml recombinant human bFGF. Human LN-metastatic prostate tumor cell lines, LNCaP, LNCaP Pro-5, and LNCaP LN-3 (28
), and human brain-metastatic prostate tumor DU-145 cells (ATCC®) were maintained in RPMI-1640 with glutamine/10% FBS/1% P/S. Human BMEC, HBMEC-60 (kindly provided by Dr. C. Ellen van der Schoot; Sanquin Research at CLB; Amsterdam, Netherlands (30
)), were cultured in Medium199 with HEPES and glutamine/10% FBS/10% human serum/100μg/ml G418/5U/ml heparin/1ng/ml recombinant human bFGF/1% P/S.
Parallel-Plate Flow Analysis.
For cell rolling assessments on E-selectin natively expressed on human BMEC, prostate tumor cells and (+) control KG1a cells were perfused over confluent cultures of HBMEC-60 grown in 35x10mm culture dishes (Corning Inc., Corning, NY) and stimulated for 4 hr with 10ng/ml IL-1β (Sigma Co., St. Louis, MO) prior to assay as previously described (20
). To confirm E-selectin expression, cells were harvested with 0.5mM EDTA and stained with anti-human E-selectin moAb 68-5H11 (BD Biosciences, Inc., San Jose, CA) for flow cytometric analysis. IL-1β-stimulated HBMEC-60 cells treated with 10μg/ml neutralizing anti-human E-selectin monoclonal Ab 68-5H11 for 30 min. at RT was performed to confirm E-selectin-mediated adhesion. Prostate tumor cells released with 0.5mM EDTA and washed twice in PBS were suspended at 1x106
cells/ml in HBSS/10mM HEPES/2mM CaCl2
assay medium and then infused into the chamber over HBMEC-60 cultures as previously described (20
). Cell rolling was assessed at 0.6 dynes/cm2
from the mid-point of the chamber viewing field (4 fields of view, 3 different experiments) at 100X magnification as previously described (31
). All experiments were observed in real time and videotaped for offline analysis.
Immunoprecipitations and Western Blot Analysis.
Membrane preparations of prostate tumor, WEHI-3 and KG1a cells were prepared, and membrane protein was quantified by Bradford method as previously described (14
). E-selectin ligand, PSGL-1 and ESL-1 were immunoprecipitated from membrane protein solubilized in 2% NP-40 and precleared in Protein G-agarose with recombinant murine E-selectin-human Ig chimera (R & D Systems, Inc., Minneapolis, MN), mouse IgG anti-human PSGL-1 moAb KPL-1 (BD Biosciences, Inc., San Jose, CA) and rabbit polyclonal anti-sera against ESL-1, respectively. ESL-1 anti-sera was generously provided by Dr. Bruce Furie (Beth-Israel Deaconess Medical Center, Boston, MA) and was also prepared by immunizing rabbits with KLH-conjugated peptides from amino acids 157-176 of ESL-1 (Invitrogen Corp., Carlsbad, CA). Solubilized membrane protein precleared in protein G-agarose was mixed with antibody/chimera at a mass ratio of 100:4, incubated for 18hr on a rotator at 4°C, mixed with protein-G agarose for 2hr at 4°C, and then analyzed by Western blotting. Immunoprecipitations performed with respective isotype controls were conducted in parallel to control for non-specific protein binding. Where indicated, surfaces of WEHI-3 and prostate tumor cells were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin (Pierce Biotechnology, Inc., Rockford, IL) according to manufacturer's protocol prior to membrane protein preparation. Biotinylated membrane protein was incubated with avidin-agarose (Vector Labs., Burlingame, CA) for 18hr on a rotator at 4°C and analyzed by Western blotting.
For Western blotting, total membrane protein, biotinylated membrane protein or avidin/immunoprecipitates were subjected to reducing SDS-PAGE on 4-20% gradient gels, transferred to Immunoblot™ PVDF membrane (Bio-Rad, Inc., Hercules, CA), and blotted with respective antibody. To confirm that quantified membrane protein was equivalent, identically-loaded SDS-PAGE gels were prepared in parallel and stained with Coomassie blue R-250. Intensity analysis of Coomassie-blue stained protein showed that membrane protein loaded for blotting experiments was identical. Blots were first blocked in FBS and then incubated with E-selectin-Ig, anti-PSGL-1 moAb KPL-1, rat IgM anti-human CLA moAb HECA-452 (BD Biosciences) or ESL-1 anti-sera (all at 1 μg/ml) for 1 hr at RT. Isotype control immunoblots were performed in parallel to evaluate non-specific protein binding. Blots were then incubated with respective alkaline phosphatase (AP)-conjugated secondary Abs (all at 1:1000) (Zymed Labs. Inc., San Francisco, CA) for 1 hr at RT and developed with AP-substrate Western Blue® (Promega; Madison, WI) as previously described (20
). These experiments were performed a minimum of 5-times.
Purification and Mass Spectrometry of E-selectin-Ig-reactive 150kDa Membrane Protein. To isolate the 150kDa protein(s), we separated MDA PCa 2b membrane protein on reducing 4-20% SDS-PAGE gradient gels and analyzed the E-selectin-Ig-reactive 150kDa protein as follows. To guide localization, excision and retention of the relevant protein, an E-selectin-Ig immunostained blot was prepared in parallel, and the stained blot was superimposed with the corresponding gel. Excised gel fragments from corresponding gels were loaded into a single well (three gel fragments/well) of a new 4-20% gradient gel and subjected to reducing SDS-PAGE. This semi-purification method was repeated 3-times, and the last excised gel fragment was digested with trypsin and analyzed by MALDI-TOF mass spectrometry (Molecular Biology Core Facility, Dana Farber Cancer Institute), and the NCBInr database was searched for possible peptide matches.
Flow Cytometry. Cells from suspension cultures or from adherent cultures harvested by 5mM EDTA were washed twice with cold PBS/2%FBS and suspended in PBS/1%FBS. MoAb HECA-452, anti-human PSGL-1 moAb PL-2 (BD Biosciences), anti-human CD44 moAb A3D8 (Sigma), ESL-1 rabbit anti-sera, anti-human E-selectin moAb 68-5H11 or appropriate isotype-matched control antibody (2μg/test) was incubated with cells for 30 min. on ice. Following two washes, cells were incubated with fluorochrome-conjugated secondary antibody for 30 min. on ice. After washing twice, flow cytometry was performed using a FACScan apparatus equipped with an argon laser tuned at 488 nm (Becton Dickinson). Cells stained with relevant isotype control antibody were subtracted from cells stained with test antibody to control for non-specific binding.
Immunohistochemical Analysis. For ESL-1 immunohistochemical analysis, we utilized tissue micro-arrays (TMA) containing 4μm sections of 2-mm cores from formalin-fixed, paraffin-embedded normal prostate tissue and prostate adenocarcinoma (Chemicon International, Inc.; Temecula, CA). Prostate tumors with a Gleason score of 2-6 were designated as low grade tumors, and tumors with a Gleason score ranging from 7-10 were designated as high grade tumors. TMA were deparaffinized and rehydrated according to manufacturer's protocol. For antigen retrieval, TMA were incubated in 10mM citrate buffer (pH 6.0) in a steam pressure cooker per manufacturer's instructions (Biocare Medical, Walnut Creek, CA). TMA were blocked in hydrogen peroxide and normal goat serum (1:20), and then incubated with rabbit anti-sera against ESL-1 (1:1000) for 1hr at RT. After washing, TMA were incubated with HRP linked-anti-rabbit IgG (Envision Plus Kit; DakoCytomation, Inc., Carpinteria, CA) for 30 min. at RT. Staining was performed using standardized development times and diaminobenzidine (Dako) as a substrate. All TMA were counterstained in hematoxylin. For semi-quantitative analysis of cell staining, brown-stained tumor cells were enumerated and divided by total tumor cell count per field of view at 200X magnification (0.785mm2) and multiplied by 100 to obtain a percent positive cell staining value. Two 2mm cores (a minimum of 4 fields of view) were examined per prostate tissue specimen. Using isotype control staining as a reference for background levels, cell staining was scored as absent (≤1% positive tumor cell staining), weak to moderate (≤75% positive tumor cell staining) and high (>75% positive tumor cell staining).
For PSGL-1 analysis, TMA were constructed and immunostained as follows:
TMA were generated from 30 rapid autopsies and represent prostate tumor specimens from the prostate gland and from all potential metastatic sites (32
). The rapid autopsy program was approved by the Institutional Review Board of The University of Michigan. TMA cores were assembled using the manual tissue arrayer (Beecher Instruments, Silver Spring, MD) as previously described (33
). Tissue cores from designated areas were targeted for transfer to array blocks. Tissue cores in triplicate (0.6mm in diameter) were sampled from each representative tissue block and spaced at 0.8mm from core-center to core-center. After construction, 4 μm sections were cut and stained with hematoxylin and eosin to verify histological diagnosis. All data are maintained on a relational database as previously described (34
). TMA were deparaffinized and rehydrated and then subjected to antigen retrieval in 10mM citrate buffer (pH 6.0) in a pressure cooker for 2 minutes. For immunostaining, TMA were blocked in hydrogen peroxide and normal goat serum (1:20), and then incubated with mouse anti-human PSGL-1 antibody (clone PL-2) (1:200) for 1hr at RT. After washing, TMA were incubated with HRP-linked anti-murine IgG (Envision Plus Kit; Dako), developed and counterstained as described above.
Semi-Automated Quantitative Image Analysis. A semi-automated quantitative image analysis system, ACIS II (Chromavision, San Juan Capistrano, CA), was used to evaluate stained TMA. For immunohistochemical analysis, proprietary software for the ACIS II device was used to detect and quantify brown staining intensity and then compares this value to blue counterstain representing background. Theoretical intensity levels range from 0-255 chromogen intensity units (CIU). In pilot experiments for this study, reproducibility of the ACIS II system was tested and confirmed by scoring several TMA. The correlation coefficient for these experiments was r2=0.973. Due to tissue heterogeneity and to the potential of false-positive leukocyte staining with anti-PSGL-1, a pathologist electronically circled the areas of interest on each tissue core with the ACIS II software, ensuring that intensity measurements were consistent with selected stained areas. Benign prostate tissue cores were included on each TMA to help normalize each slide.
Intensity values were obtained for each tissue core and then normalized within each TMA. Due to potential variation of immunohistochemical staining and intensity values for each TMA, data were normalized for each TMA and from multiple TMA. Staining intensity values for each tissue core from a given TMA were subtracted by the mean intensity for that same TMA and then divided by the standard deviation:
= 1, …, ni ( ni
is the total number of cores on TMAi )
. As a result, each normalized TMA had a mean score of 0 and standard deviation equal to 1. Data were then combined using this normalized scale and analyzed using the SPSS software (SPSS Systems, Chicago, IL).