Title: Formation of Hyaluronan- and Versican-rich Pericellular Matrix by Prostate Cancer Cells Promotes Cell Motility
Abstract: Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility. Previous studies have demonstrated that high levels of hyaluronan (HA) and the chondroitin sulfate proteoglycan, versican in the peritumoral stroma are associated with metastatic spread of clinical prostate cancer. In vitro integration of HA and versican into a pericellular sheath is a prerequisite for proliferation and migration of vascular smooth muscle cells. In this study, a particle exclusion assay was used to determine whether human prostate cancer cell lines are capable of assembling a pericellular sheath following treatment with versican-containing medium and whether formation of a pericellular sheath modulated cell motility. PC3 and DU145, but not LNCaP cells formed prominent polarized pericellular sheaths following treatment with prostate fibroblast-conditioned medium. The capacity to assemble a pericellular sheath correlated with the ability to express membranous HA receptor, CD44. HA and versican histochemical staining were observed surrounding PC3 and DU145 cells following treatment with prostatic fibroblast-conditioned medium. The dependence on HA for integrity of the pericellular sheath was demonstrated by its removal following treatment with hyaluronidase. Purified versican or conditioned medium from Chinese hamster ovary K1 cells overexpressing versican V1, but not conditioned medium from parental cells, promoted pericellular sheath formation and motility of PC3 cells. Using time lapse microscopy, motile PC3 cells treated with versican but not non-motile cells exhibited a polar pericellular sheath. Polar pericellular sheath was particularly evident at the trailing edge but was excluded from the leading edge of PC3 cells. These studies indicate that prostate cancer cells recruit stromal components to remodel their pericellular environment and promote their motility. Versican is a large chondroitin sulfate proteoglycan (CSPG) 2The abbreviations used are: CSPG, chondroitin sulfate proteoglycan; BrdU, bromodeoxyuridine; BSA, bovine serum albumin; CM, conditioned medium; CS, chondroitin sulfate; ChABC, chrondroitinase ABC; ECM, extracellular matrix; FBS, fetal bovine serum; GAG, glycosaminoglycan; HA, hyaluronan; HABP, hyaluronan-binding protein; Hase, hyaluronidase; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay. consisting of a core protein (Mr > 400 kDa) with 12-15 chondroitin sulfate (CS) side chains covalently attached. It belongs to a family of extracellular proteoglycans (hyalectins) that bind to hyaluronan (HA) of which aggrecan, the cartilage-specific proteoglycan is the prototype (1LeBaron R.G. Perspect. Dev. Neurobiol. 1996; 3: 261-271PubMed Google Scholar). Four versican isoforms have been identified. V0, V1, and V2 result from alternative splicing of two large exons each encoding a glycosaminoglycan (GAG) attachment domain (GAGα and GAGβ). V3 lacks these domains and is devoid of GAG side chains. V0 and V1 are the principal isoforms present in the interstitial matrix of most tissues. All versican isoforms contain globular domains at the N terminus (G1) and C terminus (G3). The G1 domain contains two tandem repeat link modules, which bind HA, and the G3 domain consists of a set of lectin-, epidermal growth factor-, and complement-binding protein-like subdomains with structural similarity to the selectin family (1LeBaron R.G. Perspect. Dev. Neurobiol. 1996; 3: 261-271PubMed Google Scholar, 2Yamagata M. Shinomura T. Kimata K. Anat. Embryol. (Berl). 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar). We have previously demonstrated that elevated levels of versican in peritumoral stroma, is an indicator for disease relapse following surgery for clinically localized prostate (3Ricciardelli C. Mayne K. Sykes P.J. Raymond W.A. McCaul K. Marshall V.R. Tilley W.D. Skinner J.M. Horsfall D.J. Clin. Cancer Res. 1997; 3: 983-992PubMed Google Scholar, 4Ricciardelli C. Mayne K. Sykes P.J. Raymond W.A. McCaul K. Marshall V.R. Horsfall D.J. Clin. Cancer Res. 1998; 4: 963-971PubMed Google Scholar, 5Ricciardelli C. Quinn D.I. Raymond W.A. McCaul K. Sutherland P.D. Stricker P.D. Grygiel J.J. Sutherland R.L. Marshall V.R. Tilley W.D. Horsfall D.J. Cancer Res. 1999; 59: 2324-2328PubMed Google Scholar) and breast cancer (6Ricciardelli C. Brooks J.H. Suwiwat S. Sakko A.J. Mayne K. Raymond W.A. Seshadri R. LeBaron R.G. Horsfall D.J. Clin. Cancer Res. 2002; 8: 1054-1060PubMed Google Scholar, 7Suwiwat S. Ricciardelli C. Tammi R. Tammi M. Auvinen P. Kosma V.M. LeBaron R.G. Raymond W.A. Tilley W.D. Horsfall D.J. Clin. Cancer Res. 2004; 10: 2491-2498Crossref PubMed Scopus (123) Google Scholar). Our more recent studies demonstrated that purified versican from cultured human prostatic fibroblasts inhibited adhesion of prostate cancer cells to a fibronectin substratum in vitro (8Sakko A.J. Ricciardelli C. Mayne K. Suwiwat S. LeBaron R.G. Marshall V.R. Tilley W.D. Horsfall D.J. Cancer Res. 2003; 63: 4786-4791PubMed Google Scholar). These findings are consistent with versican modulating tumor cell attachment to the stromal matrix in vivo, a primary facet controlling cancer cell motility and invasion. CSPGs such as versican interact with other extracellular matrix (ECM) molecules and form macromolecular complexes (9Kohda D. Morton C.J. Parkar A.A. Hatanaka H. Inagaki F.M. Campbell I.D. Day A.J. Cell. 1996; 86: 767-775Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The expression of CD44 on the cell membrane of chondrocytes is essential to direct assembly of HA pericellular matrix (10Knudson W. Knudson C.B. J. Cell Sci. 1991; 99: 227-235Crossref PubMed Google Scholar). Urinary bladder carcinoma cell lines that express CD44 receptors but minimal HA and versican have also been reported to assemble prominent pericellular matrices following the addition of exogenous HA and aggrecan (10Knudson W. Knudson C.B. J. Cell Sci. 1991; 99: 227-235Crossref PubMed Google Scholar). Evanko et al. (11Evanko S.P. Angello J.C. Wight T.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1004-1013Crossref PubMed Scopus (417) Google Scholar) recently reported that an HA coat incorporating versican is obligatory for the proliferation and migration of smooth muscle cells in vitro. Participation of versican core protein in matrix assembly through its HA binding property, and its numerous highly negatively charged CS side chains might be integral to modulation of cellular adhesion and enhancement of motility and invasion. In this study we investigated whether prostate cancer cells can utilize the ECM components, HA and versican, secreted by prostatic fibroblasts to assemble a pericellular matrix and promote cancer cell motility. Cell Culture and Collection of Prostatic Fibroblast-conditioned Medium—The human prostate adenocarcinoma cell lines LNCaP, PC3, and DU145 and Chinese hamster ovary (CHO K1) cell lines were purchased from the American Type Culture Collection. CHO K1 cells overexpressing recombinant V1 (rV1) versican (CHO V1) were kindly provided by Prof. R. LeBaron (Division of Life Science, University of Texas at San Antonio). The prostate cancer cell lines were maintained in 80 cm2 flasks in complete RPMI 1640 medium (Invitrogen) supplemented with 4 mml-glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin, 2 μg/ml amphotericin B, and 5% FBS. The CHO K1 and CHO V1 were maintained as described for the prostate cancer cells but using α-MEM nucleoside-free medium (JRH Biosciences, Lenexa, KS) as described previously (12LeBaron R.G. Zimmermann D.R. Ruoslahti E. J. Biol. Chem. 1992; 267: 10003-10010Abstract Full Text PDF PubMed Google Scholar). Fibroblasts were isolated from prostatic tissue obtained with informed consent from patients undergoing transurethral resection of the prostate to resolve the urine voiding symptoms of benign prostatic hyperplasia (8Sakko A.J. Ricciardelli C. Mayne K. Suwiwat S. LeBaron R.G. Marshall V.R. Tilley W.D. Horsfall D.J. Cancer Res. 2003; 63: 4786-4791PubMed Google Scholar). The prostate fibroblasts were maintained in RPMI containing 5% FBS and were shown to be free of Mycoplasma by PCR analysis. Prostate fibroblasts, CHO K1 or CHO V1 were plated at a density of 1 × 104 cells/cm2 and incubated in culture medium for 72 h prior to collecting conditioned medium (CM). The CM was used fresh or stored at -70 °C for up to 12 months before use. Versican Purification and Western Immunoblotting—Versican was isolated from prostate fibroblast or CHO V1 CM (recombinant versican, rV1) using a combination of anion exchange and gel filtration chromatography. Briefly, CM stored at -70 °C was thawed and batch-adsorbed to Q-Sepharose (10 ml/liter of medium, Amersham Biosciences, Uppsala, Sweden) at 4 °C for 24-48 h in the presence of protease inhibitor tablets (Roche Applied Science), batch-eluted with 2 m NaCl in PBS, pH 7.4 and purified by gel chromatography on a Sephacryl S400 (15 × 650-mm column, Amersham Biosciences) as described previously (8Sakko A.J. Ricciardelli C. Mayne K. Suwiwat S. LeBaron R.G. Marshall V.R. Tilley W.D. Horsfall D.J. Cancer Res. 2003; 63: 4786-4791PubMed Google Scholar). Versican-containing fractions were concentrated 10-20-fold using Centriprep centrifugal filters (Amicon Bioseparations, Bedford, MA) and Nanosep™ microconcentrators (Pall Gelman Laboratory, Ann Arbor, MI) with Mr 50,000 and 300,000 cut-offs, respectively. The molecular integrity of the purified versican samples was determined by immunoblotting with the rabbit antibody to recombinant human versican (Vc) provided by Prof. LeBaron as previously described (13du Cros D.L. LeBaron R.G. Couchman J.R. J. Investig. Dermatol. 1995; 105: 426-431Abstract Full Text PDF PubMed Scopus (111) Google Scholar). To determine if other CS proteoglycans were co-purified with versican, a parallel membrane was incubated with anti-chondroitin sulfate mouse monoclonal antibody (2B6, C-4-S, ICN Biochemicals, Aurora, OH). Visualization was achieved by anti-rabbit IgG or anti-mouse IgG peroxidase-conjugated secondary antibodies (Dako Labs, Glostrup, Denmark) and enhanced chemiluminescence (ECL, Amersham Biosciences). Gels run in parallel were also stained using Coomassie Blue (Difco Laboratories, Surrey, UK) and silver staining (Bio-Rad) to assess the purity of the versican fractions. Results from a typical versican purification are shown in Fig. 1. The highest concentration of versican was found in pooled fractions 9 + 10 (isoforms V0 + V1, ∼400 kDa, Fig. 1A). A parallel gel immunoblotted with a CS monoclonal antibody demonstrated a broad band at ∼400 kDa, consistent with the versican, and an additional minor band at ∼150 kDa (Fig. 1B). This 150-kDa band was present in the versican immunoblot and is likely to be a versican degradation product. These bands but no other contaminating protein bands were observed in the silver stained gel (Fig. 1C). No contaminating bands were observed in fractions 7 + 8 or fractions 9 + 10 in a parallel gel stained with Coomassie Blue (not shown). Versican Quantitation—As versican levels in purified fractions from prostatic fibroblast CM were too low to be accurately determined by colorimetric protein assays, a versican ELISA was developed. In the absence of a quantified versican standard being available, the versican concentration was determined relative to a highly purified and concentrated fraction of rV1 versican defined as containing 100 arbitrary units/ml. Versican samples (50 μl) diluted in PBS containing 0.1% BSA and 0.05% Tween 20 were bound to 96-well tissue culture plates for 2 h at room temperature in PBS and washed with PBS/0.05% Tween 20 prior to the addition of versican monoclonal antibody (12C5, 1/250, developed by Dr. Richard Asher and obtained from by the Developmental Studies Hybridoma Bank, NICHD, University of Iowa). Following an overnight incubation at 4 °C, the wells were washed three times with PBS/0.05% Tween 20 and incubated with 100 μl of anti-mouse IgG peroxidase-conjugated secondary antibody (1/2000) for 90 min at room temperature. Wells were subsequently washed three times with PBS/0.05% Tween 20. Substrate solution (100 μl, 0.4 mg/ml o-phenylenediamine in 0.01% H2O2, 0.1 m citrate phosphate buffer, pH 5.0) was added to each well and after 15 min at room temperature, the reaction was stopped by addition of 25 μl of 2.5 m H2SO4 and absorbance was read at 450 nm. Negative controls included no primary antibody and no secondary antibody and CHO K1 CM and aggrecan instead of versican. A typical standard curve in the versican ELISA had the equation y = 0.054x + 0.039 with an R2 = 0.986. Relative versican concentrations ranged between 0.1-0.4 units/ml versican in prostatic fibroblast CM and 1.0-5.0 units/ml versican in the purified versican fractions from prostatic fibroblast CM. Quantitation of HA Synthesis—The concentration of HA in cell culture supernatants was determined by a competitive binding assay (14Simpson M.A. Reiland J. Burger S.R. Furcht L.T. Spicer A.P. Oegema Jr., T.R. McCarthy J.B. J. Biol. Chem. 2001; 276: 17949-17957Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Tissue culture microtiter plates (96 well) were coated with human umbilical cord HA (Sigma-Aldrich) at 50 μg/ml in 200 mm sodium carbonate buffer (pH 9.6) for 4 h at 37 °C. Unbound HA was removed with four washes of PBS/0.05% Tween 20. CM (48 h) was harvested from prostate carcinoma cells (PC3, DU145, and LNCaP) or prostate fibroblasts plated (2 × 104/well) cultured in 5% FBS RPMI in 12-well plates. Final cell numbers were determined after trypsinization by manual counting using a hemacytometer. CM from prostate cells (100 μl) was combined with 100 μl of biotinylated HA-binding protein (1 μg/ml Seikagaku Corp., Tokyo, Japan) and incubated in the HA-precoated wells overnight at room temperature. The plate was washed four times with PBS/Tween 20, developed using a streptavidin HRP-system (Dako Labs) using o-phenylenediamine (Sigma-Aldrich) as a substrate, and the absorbance measured at 490 nm. The mean HA concentration for each sample was interpolated from an HA standard curve performed in parallel and normalized to cell number. Data are presented as μg of HA per 106 cells. Visualization of Pericellular Matrix—Pericellular sheath formation was visualized by a particle exclusion assay utilizing the steric exclusion property of the HA gel (11Evanko S.P. Angello J.C. Wight T.N. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1004-1013Crossref PubMed Scopus (417) Google Scholar). Prostate cancer cells (PC3, DU145, and LNCaP) were plated (3 × 103) in 48-well tissue culture plates in 5% FBS RPMI. After 48 h of culture, prostate cancer cells were treated with either prostatic fibroblast CM (0.1-0.4 units/ml versican), purified human versican from prostatic fibroblasts (0.5 unit/ml) rV1 (62.5 milliunits-4 units/ml), bovine aggrecan (0.1 mg/ml, Sigma-Aldrich) or control medium. After 24 h of treatment, 300 μl of a suspension of human red blood cells (107/ml, CSL, 0.1% BSA in PBS) was added to the prostate cancer cells and allowed to settle for 10 min. Prostatic fibroblasts (1 × 103) cultured in 5% FBS RPMI in parallel with the prostate cancer cells were used as positive control. Pericellular sheath formation was quantitated from digital photographs using the Video Pro image analysis system (Leading Edge P/L, Marion, South Australia). The area delimited by red blood cells and the area delimited by the cell membrane was measured to give a sheath to cell ratio. A ratio of 1.0 indicates no matrix. Cells with ratio of ≥2.0 were considered to have a pericellular sheath. To demonstrate the dependence on HA for the formation of the pericellular sheath, cells were subsequently treated with Streptomyces hyaluronidase (Hase, 10 units/ml, Sigma-Aldrich) for 20 min at room temperature. The requirement for CS chains in the formation of pericellular sheath was examined by preincubation of fibroblast CM with chondroitinase ABC (ChABC, 0.1 units/ml, Sigma-Aldrich) for 2 h at 37 °C. ChABC activity was subsequently inactivated by treating CM at 95 °C for 5 min. Digestion of CS chains and HA by ChABC was determined by dot blot analysis using a mouse monoclonal antibody to native CS (CS-56, Sigma-Aldrich) or biotinylated hyaluronan-binding protein (HABP, 2 μg/ml, Seikagaku Corp, Japan) and visualized with anti-mouse IgG peroxidase-conjugated secondary antibodies (Dako Labs) or streptavidin-horseradish peroxidase conjugate (Dako Labs), respectively, followed by enhanced chemiluminescence (ECL, Amersham Biosciences). Detection of CD44, HA, and Versican in Pericellular Sheath—To visualize the components of pericellular sheath, prostate cancer cells (PC3, DU145, and LNCaP 5 × 103/well) were plated onto tissue culture coverslips (Thermanox, Nunc, Rosk-ilde, Denmark) in 24-well plates in 5% FBS RPMI for 48 h and treated for 24 h with 5% RPMI, fibroblast CM (0.1 units/ml versican), CHO K1 CM, CHO V1 CM (1 units/ml versican) or purified versican fractions (0.24 units/ml versican) from prostatic fibroblast CM as described for the particle exclusion assay. The coverslips were attached to microscope slides using UV-cured glue (Loctite 349, Loctite Corporation, Rocky Hill, CA) and the cells fixed sequentially in 10% PBS-buffered formalin (10 min), ice-cold methanol (2 min), and ice-cold acetone (1 min). Following extensive washing with PBS, the cells were incubated overnight with specific antibodies to CD44 (mouse monoclonal, Clone 156-3C11, 1 μg/ml, Neomarkers, Fremont, CA) or versican (rabbit polyclonal to recombinant human versican Vc, (13du Cros D.L. LeBaron R.G. Couchman J.R. J. Investig. Dermatol. 1995; 105: 426-431Abstract Full Text PDF PubMed Scopus (111) Google Scholar) diluted 1:1000) in 10% goat serum, or the biotinylated HABP (2 μg/ml). Versican and CD44 immunoreactivity were detected using biotinylated goat anti-rabbit and anti-mouse secondary antibodies (diluted 1/400, Dako), respectively. Visualization of binding was achieved with standard streptavidin (diluted 1:500, Dako Labs) immunoperoxidase reaction and diaminobenzidine tetrahydrochloride (Sigma) to yield an insoluble brown deposit. Cells were counterstained with weak hematoxylin and mounted in Depex (BDH Laboratory Supplies, Poole, England). CD44 and Bromodeoxyuridine (BrdU) Double Immunofluorescence—The relationship between CD44 expression and PC3 cell proliferation was investigated by double labeling immunofluorescence with CD44 and BrdU antibodies. PC3 cells (3 × 103 cells/well) were plated in 8-well tissue culture chamber slides (Nunclon™ Lab-Tek II Chamber slide, RS Glass Slide, Naperville, IL) in 500 μl of 5% FBS RPMI for 48 h and treated for 24 h with control medium (5% FBS RPMI, CHO K1 CM, 0.1% BSA RPMI) or versican-containing medium (fibroblast CM, CHO V1 CM, purified versican from prostatic fibroblasts) as described for the particle exclusion assay. The cells were supplemented with 20 μm BrdU for the last 4 h of treatment. The proportion of cells forming a pericellular sheath were determined by the particle exclusion assay prior to fixation for 10 min in 4% paraformaldehyde and methanol (-20 °C) for 15 min. Slides were treated with 1 m HCl for 10 min to denature DNA (15Knapp P.E. J. Histochem. Cytochem. 1992; 40: 1405-1411Crossref PubMed Scopus (23) Google Scholar) and after neutralizing in PBS, pH 7.4, cells were blocked with 5% goat serum and incubated overnight with anti-BrdU conjugated with Alexa Fluor 594 (2.5 μg/ml, Molecular Probes, Eugene, OR) and anti-CD44 (Clone 156-3C11, 2.5 μg/ml) antibodies, and then incubated for 1 h in the dark at room temperature with goat anti-mouse immunoglobulins conjugated with Alexa 488 (Molecular Probes). The nuclei were stained with Hoescht dye (5 μg/ml, Sigma) for 15 min and mounted with fluorescent mounting medium (Dako Labs). Cells were viewed with an epifluorescence microscope (BX50, Olympus Australia) and imaged using a Spot RT digital camera (Diagnostic Instruments, Sterling Heights, MI). Boyden Chamber Motility Assays—Chemotactic motility assays were performed in a modified Boyden chamber assay as described previously (16Iida J. Pei D. Kang T. Simpson M.A. Herlyn M. Furcht L.T. McCarthy J.B. J. Biol. Chem. 2001; 276: 18786-18794Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). PC3 cells (5 × 105 cells/ml) were harvested, washed with RPMI medium containing 0.1% BSA (serum-free RPMI), and treated with purified versican from prostatic fibroblast CM (pooled fractions 9 + 10 diluted to 0.4 units/ml) or aggrecan (0.1 mg/ml) ± HA (20 μg/ml) or serum-free RPMI ± HA in the absence of versican or aggrecan for 2 h on an oscillating platform (Nutator, Clay Adams, Becton Dickinson, Sparks, MD) at room temperature to enable pericellular sheath formation. An aliquot (20 μl) of these cells from each treatment were mixed with 400 μl of a suspension of human red blood cells (107/ml, 0.1% BSA in PBS) and allowed to attach for 2 h at 37 °C in the presence or absence of Hase (10 units/ml) for assessment of sheath formation. For the motility assay, the lower chambers were filled with serumfree RPMI containing fibronectin (10 μg/ml, bovine plasma, Sigma). The PC3 cells (1 × 105, 200 μl) as treated above, were added directly to the top of 12-μm pore size polyvinylpyrroli-done-free polycarbonate filters (Nucleopore, Pleasanton, CA) and allowed to migrate for 4 h at 37°C in a 5% (v/v) CO2 incubator. An additional control included treatment of prostate cells with Hase (10 units/ml) during the 4-h migration assay. The filters were stained by Diff-Quik (Baxter, Miami, FL), and migrated cells were counted by light microscopy at ×120 magnification in ten random fields. Data are expressed as percentage of control ± S.D. Wound Migration Assays and Time Lapse Photography—Wound migration assays were performed using PC3 cells (2 × 104 cells/well) cultured in 8-well chamber slides (Nuclon™ Lab-Tek II Chamber slide, RS Glass Slide) in 500 μl of 5% FBS RPMI for 3-4 days. The resulting confluent cell monolayers were wounded with a small spatula coated with parafilm, washed to remove floating cells prior to treating with CHO K1 CM or CHO V1 CM (0.5 units/ml versican). Cell migration was monitored over a 24-h treatment period using an IX 81 microscope (Olympus) equipped with a 37 °C incubator (Solent Scientific, Segensworth, UK) and aerated with 5% CO2 in oxygen. Directional movement and pericellular sheath formation were assessed by time lapse and particle exclusion assays. Cells migrating into the wounded area were imaged over a 24-h period in the presence of red blood cells (107/ml) using Analysis® software (Soft Imaging System, Johann-Krane-Weg Munster, Germany). The number of cells migrating, the direction of motion, and proportion of cells forming a pericellular sheath was assessed. To assess CD44 and HA abundance and localization in motile PC3, the cells were fixed for 10 min in 4% paraformaldehyde and methanol (-20 °C) for 15 min prior to immunofluorescence staining. Slides were blocked with 5% goat serum and incubated overnight with anti-CD44 (2.5 μg/ml) and biotinylated HABP (1 μg/ml). Cells were incubated for 1 h in the dark at room temperature with goat anti-mouse immunoglobulins conjugated with Alexa 488 and streptavidin conjugated with Alexa 594 and subsequently counterstained with Hoechst dye (5 μg/ml). The slides were mounted with fluorescent mounting medium and imaged as described above. Statistical Analysis—The Student's t test or one-way analysis of variance test and the Dunnet t post hoc test were used to determine statistical significance between control and treatment groups. All analyses were performed using SPSS 11.0 for Windows Software (SPSS Inc., Chicago, IL). Statistical significance was accepted at p < 0.05. Pericellular Sheath Formation by Cultured Prostate Cancer Cells with Prostatic Fibroblast CM—Human prostate fibroblasts produced extensive pericellular sheaths which were entirely removed by digestion with Hase (Fig. 2A). Greater than 90% of the prostatic fibroblasts displayed HA-dependent matrices, with a mean positive cell to sheath ratio of 2.8 ± 0.9. Prominent pericellular sheaths were also assembled by PC3 and DU145 prostate cancer cells following 24 h of treatment with CM from prostate fibroblasts (Fig. 2B). Pericellular sheaths were not detected around LNCaP prostate cancer cells either in the absence or presence of fibroblast CM (Fig. 2B). Approximately 17% of PC3 (mean positive cell to sheath ratio = 2.9 ± 0.9) and 14% of DU145 (mean positive cell to sheath ratio = 2.6 ± 0.9) cells displayed pericellular sheaths following treatment with fibroblast CM (0.1 units/ml versican, Fig. 3A). In comparison, less than 5% of these cells exhibited pericellular sheaths when cultured in 5% FBS RPMI (Fig. 3A). The proportion of PC3 cells assembling a pericellular sheath was reduced with increasing dilution of fibroblast CM, reaching control levels when diluted at 1:10 (Fig. 3B).FIGURE 3A, proportion of PC3 and DU145 cells expressing pericellular sheath following treatment with prostate fibroblast CM. Cells were plated as indicated in B, and the pericellular sheath was visualized by particle exclusion assay. Data represent percentage (mean ± S.D.) of cells (n = 300) with pericellular sheath from 6 determinations in three separate experiments. *, significantly different from control. B, the proportion of PC3 cells assembling a pericellular sheath decreased with decreasing concentration of fibroblast CM in a dose-dependent manner. Data represent percentage (mean ± S.D.) of cells (n = 100) with pericellular sheath from duplicate determinations in one experiment. *, significantly different from control. C, treatment of fibroblast CM with chondroitinase ABC (ChABC, 0.1 unit/ml, 2 h at 37 °C) hadno effect on pericellular sheath formation. Data represent percentage (mean ± S.D.) of cells (n = 100) with pericellular sheath from duplicate determinations in one experiment. *, significantly different from control p < 0.05. D, dot blots with chondroitin sulfate antibody (CS-56) and biotinylated HABP following digestion with ChABC (0.1 units/ml) or Hase (10 units/ml). E, dependence of pericellular sheath on HA. Structural necessity for HA within the pericellular matrix formed by PC3 cells was confirmed following Hase treatment (10 units/ml) for 20 min at room temperature. Red blood cell diameter: 7 μm. Magnification ×341. White dashes outline the cell membrane of two PC3 cells with a prominent polar pericellular sheath and matrix breakdown upon digestion with Hase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ChABC digestion of the fibroblast CM did not affect pericellular sheath formation by PC3 cells (Fig. 3C). Complete digestion of CS chains in the fibroblast CM was confirmed by dot blot using a CS antibody (Fig. 3D). The requirement of HA for assembly of the pericellular sheath by PC3 cells was demonstrated following treatment with Hase (Fig. 3E). Hase treatment completely removed detectable HA from fibroblast CM; however, the ChABC digestion conditions used did not affect HA binding (Fig. 3D). The polarity of pericellular sheath formation was assessed in photomicrographs of PC3 following versican treatment. Of the 100 cells examined with pericellular sheath, 89 cells exhibited clear asymmetry or polarity of the pericellular sheath as shown in Figs. 2B and 3E. Pericellular Sheath Formation by PC3 Cells Treated with Purified Versican—Treatment of cultured PC3 cells with purified versican from prostatic fibroblasts (pooled fractions 9 + 10 diluted to 0.4 units/ml) in the absence of exogenous HA resulted in pericellular sheath formation to the same extent as neat fibroblast CM (0.25 units/ml versican) and purified aggrecan (0.1 mg/ml) (Fig. 4A). Pericellular sheath was also formed by PC3 cells following treatment with CM from CHO cells expressing recombinant V1 (CHO V1 CM, 1 unit/ml versican) but not with parental CHO K1 CM. Pericellu