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TRANSLATIONAL AND CLINICAL RESEARCH

Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Mediate Human Stem Cell Tropism to Malignant Solid Tumors

Divisions of ^aHematology/Hematopoietic Cell Transplantation,
^bMolecular Medicine, and
^eNeurosciences, City of Hope National Medical Center and Beckman Research Institute, Duarte, California, USA;
^cDepartment of Medicine, University of British Columbia Hospital, Vancouver, British Columbia, Canada;
^dGachon University School of Medicine, Inchon, Korea

Key Words. CD87 • Hepatocyte growth factor • Interleukin-6 • Interleukin-8 • Monocyte chemoattractant protein-1 • Cell migration • Neural stem cells • Mesenchymal stem cells • Urokinase plasminogen activator • Urokinase plasminogen activator receptor • Tissue inhibitor of metalloproteinase

Correspondence: Correspondence: Margarita Gutova, M.D., Division of Hematology/Hematopoietic Cell Transplantation, City of Hope National Medical Center and Beckman Research Institute, 1500 East Duarte Road, Duarte, California 91010-3000, USA. Telephone: 626-359-8111, ext. 63613; Fax: 626-930-5416; e-mail: mgutova{at}coh.org; or Karen S. Aboody, M.D., Division of Hematology/Hematopoietic Cell Transplantation and Neurosciences, City of Hope National Medical Center and Beckman Research Institute, 1500 East Duarte Road, Duarte, California 91010-3000, USA. Telephone: 626-471-7177; Fax: 626-301-8857; e-mail: kaboody{at}coh.org

Received on February 18, 2008; accepted for publication on April 1, 2008.

First published online in STEM CELLS EXPRESS  April 10, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Human neural and mesenchymal stem cells have been identified for cell-based therapies in regenerative medicine and as vehicles for delivering therapeutic agents to areas of injury and tumors. However, the signals required for homing and recruitment of stem cells to these sites are not well understood. Urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) are involved in chemotaxis and cell guidance during normal development and are upregulated in invasive tumors. Here we provided evidence that activation of uPA and uPAR in malignant solid tumors (brain, lung, prostate, and breast) augments neural and mesenchymal stem cell tropism. Expression levels of uPAR on human solid tumor cell lines correlated with levels of uPA and soluble uPAR in tumor cell-conditioned media. Cytokine expression profiles of these tumor-conditioned media were determined by protein arrays. Among 79 cytokines investigated, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 were the most highly expressed cytokines in uPAR-positive tumors. We provided evidence that human recombinant uPA induced stem cell migration, whereas depletion of uPA from PC-3 prostate cancer cell-conditioned medium blocked stem cell migration. Furthermore, retrovirus-mediated overexpression of uPA and uPAR in neuroblastoma (NB1691) cells induced robust migration of stem cells toward NB1691 cell-conditioned media, compared with media derived from wild-type NB1691 cells. We conclude that expression of uPA and uPAR in cancer cells underlies a novel mechanism of stem cell tropism to malignant solid tumors, which may be important for development of optimal stem cell-based therapies.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Migration of endogenous and exogenous neural stem cells (NSCs) and mesenchymal stem cells (MSCs) to areas of pathology is a critical step in tissue regeneration [1]. NSCs and MSCs have been shown to home to areas of brain pathology such as ischemic and neoplastic lesions [2, 3]. This inherent homing ability of stem cells makes them useful for regeneration of damaged tissues, as well as for targeted delivery of therapeutic substances to sites of pathology [4].

Directed cell migration is initiated in response to chemoattractants. Numerous cytokines, growth factors, and their receptors have been shown to affect stem cell migration under normal and pathological conditions. Such cytokine/receptor pairs include stromal cell-derived factor SDF-1/CXCR4 [5, 6], stem cell factor (SCF)/c-Kit [7], hepatocyte growth factor (HGF)/c-Met [8, 9], vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) [10], monocyte chemoattractant protein-1 (MCP-1)/CCR2 [11], and high mobility group box 1 (HMGB1)/RAGE [12, 13]. Among adhesion molecules, β1- and β2-integrins and L-selectin play a significant role in the mobilization and homing of stem cells [14–16]. Extracellular matrix proteins have also been associated with NSC-glioma tropism [17]. However, the molecular mechanisms of NSC and MSC migration to tumors of various origins and phenotypes are not well defined.

Urokinase plasminogen activator receptor (uPAR), also known as CD87, was first cloned in 1985 [18]. It was believed that the only role of uPAR was binding its ligand, urokinase plasminogen activator (uPA), leading to plasminogen activation and degradation of the extracellular matrix. However, recent studies have shown that interaction of uPAR with receptors of the integrin family, G-protein-coupled receptors, and vitronectin initiates activation of several intracellular signal transduction pathways involved in cell migration, adhesion, proliferation, and apoptosis [19–21]. uPA and uPAR also play a crucial role in early stages of interneuron development, which has been shown to be impaired by the absence of uPAR-mediated signaling in uPAR(–/–) knockout mice [22].

uPA and uPAR are upregulated in tumors of various origins, where they play a critical role in the development of invasive and chemoresistant cancer phenotypes [23–25]. Induction of biosynthesis of uPAR and its shedding as soluble urokinase plasminogen activator receptor (suPAR) have also been observed in acute and chronic inflammatory conditions [26]. suPAR is involved in the mobilization and migration of stem and inflammatory cells from the bone marrow to sites of injury, following granulocyte colony-stimulating factor (G-CSF) stimulation [16, 27].

Given the pleiotropic role of uPA and uPAR in cancer cell and stem cell mobility, we hypothesized that expression of uPAR on tumor cells may determine the degree of tropism of NSCs and MSCs to solid tumors of various origins. Here we report that expression of uPAR in human cancer cell lines leads to release of uPA and suPAR into tumor cell-conditioned media. Cell migration assays revealed that chemoattraction of NSCs and MSCs to cancer cells strongly correlated with uPAR expression levels on tumor cells. We determined the cytokine profiles of conditioned media derived from high and low uPAR-expressing tumor cells. Expression of uPAR on cancer cells was associated with elevation of numerous cytokines, including interleukin (IL)-6, IL-8, MCP-1, and HGF, in the tumor-conditioned media. In summary, our results demonstrate that uPA and uPAR are significant mediators of stem cell tropism to tumors.

	MATERIALS AND METHODS

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Immunocytochemistry and Flow Cytometry Analysis
Human cancer cell lines derived from brain (U251), neuroblastoma (SK-N-AS, NB1691), lung (H1415, H211, H1915), prostate (PC-3), colon (COLO-320), and breast (MDA-MB-231, MCF-7) were obtained from American Type Culture Collection ([ATCC] Manassas, VA, http://www.atcc.org). U251, SK-N-AS, NB1691 MDA-MB-231, and MCF-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Irvine Scientific, Santa Ana, CA, http://www.irvinesci.com), and H1415, H211, COLO-320, and H1915 were grown in RPMI 1640 modified medium (ATCC), both supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 units/ml penicillin, 100 µg/ml streptomycin at 37°C in 6% CO₂. The PC-3 cell line was grown in Ham's F-12K medium with 2 mM L-glutamine supplemented with 10% FBS (ATCC). For flow cytometry analysis, each cell line was detached by trypsinization and resuspended in staining buffer (SB) (Hanks' balanced saline solution [HBSS]; Irvine Scientific, Santa Ana, CA, http://www.irvinesci.com) supplemented with 2% FBS and 10 mM HEPES at a density of 5 x 10⁶ cells per milliliter. Fifty microliters of cells (2.5 x 10⁵ cells) was added to each well of a 96-well V-shaped plate. Antibodies (fluorescein isothiocyanate [FITC]-conjugated uPAR) were added in individually titrated concentrations (10 µl per 10⁶ cells). The 96-well plates were placed on ice, and cells were incubated with antibodies for 30 minutes in the dark. Then, 150 µl/well of wash buffer (HBSS, supplemented with 15% FBS and 10 mM HEPES) was added, and the plates were centrifuged at 500g for 5 minutes at 4°C. The cell pellets were resuspended in SB supplemented with propidium iodide (1 µg/ml) to exclude nonviable cells and subjected to flow cytometric analysis. Alternatively, cells were cultured on glass slides with a Teflon barrier (Electron Microscopy Sciences, Hatfield, PA, http://www.emsdiasum.com) overnight, fixed in 4% paraformaldehyde, washed in phosphate-buffered saline, and incubated in blocking solution (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) for 1 hour at room temperature. Cells were then stained with FITC-conjugated anti-uPAR monoclonal antibodies (American Diagnostica Inc., Stamford, CT, http://www.americandiagnostica.com) at a 1:100 dilution in antibody diluent (DakoCytomation) overnight at 4°C. Slides were washed and incubated with secondary antibody (biotinylated anti-mouse IgG at 1:250 dilution) for 1 hour at room temperature. Slides were washed, stained with avidin-FITC (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) at a 1:1,000 dilution, and incubated for 1 hour. Thereafter, the slides were counterstained with 4,6-diamidino-2-phenylindole and examined using fluorescent microscopy. In all staining experiments, negative controls included omission of the primary antibodies and staining with isotype-matched mouse IgG.

Real-Time Polymerase Chain Reaction
Total RNA was isolated from cancer cell lines by using the Qiagen (Hilden, Germany, http://www1.qiagen.com) RNeasy kit according to the manufacturer's recommendations. Standard real-time polymerase chain reaction (PCR) was performed using the following primers: β-actin, 5'-GCC GAT CCA CAC GGA GTA CT-3' (forward), 5'-CTG GCA CCC AGC ACA ATG-3' (reverse); uPAR, 5-GCC CAA TCC TGG AGC TTG A-3' (forward), 5'-TCC CCT TGC AGC TGT AAC ACT-3' (reverse); and uPA, 5'-TTG CTC ACC ACA ACG ACA TT-3' (forward), 5'-GGC AGG CAG ATG GTC TGT AT-3' (reverse).

Culturing of HB1.F3 Neural Stem Cells
The HB1.F3 cell line is a multipotent, cloned cell line that was generated by immortalizing cells obtained from the telencephalon of a human fetus of 15 weeks' gestation, using a retrovirus encoding the v-myc gene. The primary cells were obtained in accordance with the guidelines of the Anatomical Pathology Department of Vancouver General Hospital, with permission to use fetal tissue granted by the Clinical Research Screening Committee Involving Human Subjects of the University of British Columbia. HB1.F3 is an established, well-characterized, stable cell line [28, 29]. HB1.F3 cells are nontumorigenic and multipotent and can be induced to differentiate into neurons, oligodendrocytes, and astrocytes. The use of this cell line circumvented the significant problem of limited availability of large quantities of primary cells and maximized reproducibility among experiments. Human NSCs (HB1.F3 and HB1.F5; two immortalized clonal lines, derived from the same human fetal telencephalon) were grown in DMEM supplemented with 10% FBS at 37°C in 6% CO₂. Multiple migration assays confirmed that both HB1.F3 and HB1.F5 NSCs display similar migration properties.

Culturing of Mesenchymal Stem Cells
Human fetal bones were dissected from 18–24-week-old fetuses obtained by elective abortion (Advanced Bioscience Resources, Alameda, CA). Tissues were obtained with approved consent of Institutional Review Board (IRB number 94014). Mononuclear cells were isolated from fetal bone marrow using Ficoll gradient centrifugation. Briefly, femurs of elective abortion fetuses were rinsed with HBSS, and bones were trimmed of muscle and fat tissue. The center of the marrow was scraped with blunt forceps, collected, washed, and passed through nylon mesh. The collected bone marrow cells were purified for mononuclear cells via Ficoll density gradient fractionation. MSCs were cultured in growth medium consisting of DMEM 1% glucose, 15% heat-inactivated FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37°C in 6% CO₂. After the third passage, the resulting cells were analyzed and found to react with SH2, SH3, and SH4 antibodies, which detect CD105 (endoglyn) and CD73, two antigens coexpressed on MSCs. The cells were then further characterized for MSC phenotype.

Migration Assay
In vitro chemotaxis assays were conducted using 24-well cell culture plates with polycarbonate inserts (Millipore, Billerica, MA, http://www.millipore.com) with pore diameters of 8 or 10 µm for NSCs and MSCs, respectively. In brief, conditioned media were prepared by addition of serum-free media to cultured cells (75% confluence), followed by incubation at 37°C, 6% CO₂ for 48 hours. Conditioned media from tumor lines were collected and added to the lower chamber of 24-well plates (600 µl). Inserts were placed into wells, and a suspension of stem cells was added in the upper chamber (10⁵ cells per 400 µl in DMEM supplemented with 2% bovine serum albumin [BSA]). After incubation of the plates for 4 hours at 37°C, the cells that did not migrate were removed from the inner surface of the filter, whereas migrated cells were detached from the lower surface of the insert by trypsinization. Detached cells were centrifuged for 5 minutes and counted using the Guava ViaCount assay (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com). Only viable cells were included in our data analysis. The Guava ViaCount assay distinguishes between viable and nonviable cells on the basis of the differential permeability of DNA-binding dyes in the ViaCount reagent, and therefore, fluorescence of the dyes allows the quantitative assessment of both viable and nonviable cells in suspension. Migration assay controls were as follows: as a negative control, stem cells resuspended in 5% BSA were added to the upper chamber, with 5% BSA in the bottom chamber; as a positive control, 10% FBS was added to the lower chamber as a chemoattractant.

Cells were allowed to migrate to human recombinant uPA (catalog no. 124 HMW-uPA; American Diagnostica) after addition of 0.25–100 ng/ml of uPA to the lower chamber in serum-free media, and the migration assay was performed under same conditions as described above. Inhibition of HB1.F3 neural stem cell migration was achieved by preincubation of stem cells with uPAR function-inhibiting antibodies (1 hour, 1 µg per 10⁵ cells, 37°C), and cells were applied to migration assay. Small interfering RNAs (siRNAs) were designed to inhibit expression of uPAR in PC-3 cells using TriFECTa Dicer-Substrate RNAi system (IDT Inc., Coralville, IA, http://www.idtdna.com). Three siRNAs against human uPAR were designed using IDT RNAi Design Software and purchased from IDT (NM_002659 [GenBank] .3.1, NM_002659 [GenBank] .3.4, NM_002659 [GenBank] .3.8). Briefly, PC-3 cells were plated at a density of 2 x 10⁴ cells per well in a 24-well plate. After 24 hours, cells were transfected using 10, 1, or 0.1 nM siRNA duplexes. Hypoxanthine guanine phosphoribosyltransferase 1 dicer-substrate (HPRTS1 DS) positive control and scrambled negative control duplexes were used at concentration of 10 nM. X-tremeGENE siRNA transfection reagent was used for all transfections (catalog no. 04476093001; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Optimal inhibition of uPAR expression was achieved by using transfection of 10 nM plasminogen activator urokinase receptor-siRNA. Cells were analyzed for uPAR expression by flow cytometry and reverse transcription (RT)-PCR after 48 hours of transfection. Cell numbers were normalized, and cells were plated for collection of conditioned media at 48 hours; these media were used for migration assays.

Depletion of uPA from PC-3 cell-conditioned media was achieved by incubation of media with anti-uPA antibody (isotype-matched mouse IgG as control) bound to protein A-coated Sepharose beads (18 hours, 4°C). Stem cell migration assay was performed using uPA-depleted conditioned media.

uPA and uPAR Enzyme-Linked Immunosorbent Assay
Immunobind uPA and uPAR enzyme-linked immunosorbent assays (ELISAs) were performed according to the manufacturer's protocol (American Diagnostica). In brief, tumor cell-conditioned media were incubated in microtest wells that were precoated with uPA or suPAR. uPA ELISA recognizes uPA (single chain and pro-uPA) and uPA/plasminogen activator inhibitor-1 (PAI-1) complexes, whereas suPAR ELISA recognizes suPAR and suPAR/PAI-1 complexes. Biotinylated secondary antibodies that recognized the primary antibodies bound to uPA and uPAR molecules were then added. Addition of streptavidin-conjugated horseradish peroxidase (HRP) completed the formation of antibody-enzyme complexes. 3.3', 5.5'-tetramethylbenzidine substrate was added to create a color reaction. The reaction was stopped by addition of 0.5 M sulfuric acid, and solution absorbance was measured at 450 nm. Absorbance values were compared with those of a standard curve.

Cytokine Profiling of Tumor-Conditioned Media
Cytokine profiles of tumor cell-conditioned media were detected by the Cytokine Antibody Array V (RayBiotech, Norcross, GA, http://www.raybiotech.com) according to the manufacturer's instructions. Briefly, nitrocellulose blots were blocked for 1 hour and then incubated overnight at 4°C with undiluted conditioned medium derived from tumor cells. Blots were incubated at room temperature with a 1:500 dilution of biotin-conjugated antibodies for 2 hours, washed, and incubated for 1 hour at room temperature with a 1:10,000 dilution of HRP-conjugated streptavidin. Chemiluminescent detection of captured cytokines was quantified with the EPI Chemi II kit (RayBiotech), and films were analyzed using NIH ImageJ software. Signal intensity at each pixel was determined, and the relative intensity of each spot on the array was quantified by summing the intensities of each pixel within the spot. For each array, negative control intensities were used to determine background, which was subsequently subtracted. Resulting data were normalized to the average of four positive control spots to yield a final normalized value for each cytokine. Inducers and noninducers of stem cell migration were grouped and averaged together for statistical comparison. Relative expression of the cytokine levels in the conditioned media from the cell lines was graphically represented.

Overexpression of uPA and uPAR in NB1691 Cells
The uPA-pBABE and uPAR-pBABE constructs were obtained from Cell Biolabs, Inc. (San Diego, http://www.cellbiolabs.com). Retroviruses containing uPA and uPAR were generated using the Pantropic Retroviral Expression System (Clontech, Palo Alto, CA, http://www.clontech.com). Briefly, GP2-293 packaging cells were plated at a density of 4 x 10⁶ cells per 150-mm cell culture plate overnight and then transfected with the constructs of interest. The culture medium was collected after 48 hours and filtered through a 0.45-µm syringe-mounted filter (Fisher, Hampton, NH, http://www.fishersci.com). The medium was then concentrated using a Sorvall 90SE ultracentrifuge at 25,000 rpm at 4°C for 1.5 hours. Concentrated retroviral supernatant was used to transduce 0.5 x 10⁶ NB1691 cells per 35-mm plate over a 24-hour period. Stable lines were then selected using 2 µg/ml puromycin and/or 100 µg/ml hygromycin. Colonies were selected and screened for transgene expression using real-time PCR.

	RESULTS

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uPAR Expression in Tumor Cell Lines
We used flow cytometric analysis and immunocytochemistry to detect uPAR expression in cell lines derived from human prostate, lung, breast, colon, and brain cancers (Fig. 1A, 1C). On the basis of the expression levels of uPAR, the cell lines were grouped as follows: (a) cells expressing high levels of uPAR (>20% of cells uPAR-positive), or (b) cells expressing low levels of uPAR (<5% of cells uPAR-positive). The cell lines with high uPAR expression were derived from invasive high-grade human tumors such as glioblastoma multiforme (U251), neuroblastoma (SK-N-AS), breast carcinoma (MDA-MB-231), prostate cancer (PC-3), and non-small-cell lung cancer (H1915). The cell lines expressing low levels of uPAR were derived from less aggressive tumors of colon (COLO 320), breast (MCF-7), metastatic neuroblastoma (NB1691), and small-cell lung (H1415, H211) cancer. The uPA and uPAR expression levels were confirmed by quantitative real-time RT-PCR (Fig. 1B). We also performed immunocytochemical analysis of uPAR in cultured cells, which indicated expression of uPAR on the cell surface and in the cytoplasm of tumor cells, validating the flow cytometry data (Fig. 1C).

View larger version (30K): [in this window] [in a new window]   Figure 1. uPAR expression in cell lines derived from prostate, lung, breast, colon, and brain cancers using flow cytometry analysis (A), real-time quantitative reverse transcription-polymerase chain reaction (B), and immunofluorescent staining (C). On the basis of expression levels of uPAR, the cell lines were grouped as (a) cell lines expressing high levels of uPAR (>20%), or (b) cell lines expressing low levels of uPAR (<5%). Green staining represents uPAR immunofluorescence; blue staining is 4,6-diamidino-2-phenylindole nuclear stain (C). Abbreviations: uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor.

View larger version (27K): [in this window] [in a new window]   Figure 2. In vitro migration assay of a human neural stem cell line (HB1.F3) and hfMSCs. Neural stem cells (NSCs) showed a robust migration to conditioned media derived from tumor cells with high levels of urokinase plasminogen activator receptor (uPAR) (SK-N-AS, U251, PC-3, MDA-MB-231, and H1915) (A), whereas we detected little migration to conditioned media from tumor cells with low uPAR expression (COLO-320, NB1691, H1415, H211, and MCF-7) (A). hfMSCs showed a similar migration pattern compared with NSCs (B). Data are expressed as mean ± SD of triplicate measurements. The figure represents the pooled data from three independent experiments. Abbreviations: BSA, bovine serum albumin; FBS, fetal bovine serum; hfMSC, fetal human bone marrow-derived primary mesenchymal stem cell.

View larger version (18K): [in this window] [in a new window]   Figure 3. Upregulation of uPA receptor on tumor cell lines is associated with secretion of high levels of uPA (A) and increased uPA receptor shedding (suPAR) into the conditioned media (B). Abbreviations: suPAR, soluble urokinase plasminogen activator receptor; uPA, urokinase plasminogen activator.

View larger version (56K): [in this window] [in a new window]   Figure 4. Cytokine expression profile of the CM derived from tumor cell lines. (A): Cytokine array of CM derived from various cancer cell lines. (B): Key for the cytokine array. (C): Graphical representation of the relative expression of cytokines in the CM derived from cancer cell lines. Films were scanned and analyzed using ImageJ software. The average values for migrating versus nonmigrating cell lines were calculated and used for statistical comparison. Paired t test for each cytokine was performed to compare the mean values of cytokine expression in CM from the group of uPAR(+) versus uPAR(–) cancer cell lines. The group of uPAR(+) cell lines showed overall higher expression of cytokines than the group of uPAR(–) cell lines, which was statistically significant (*, p= .004; t= 3.556; df= 11). Abbreviations: BDNF, brain-derived neurotrophic factor; CM, conditioned media; GCSF, granulocyte colony-stimulating factor; HGF, hepatocyte growth factor; IL, interleukin; LIF, leukemia inhibitory factor; MCP, monocyte chemoattractant protein; MCSF, macrophage colony-stimulating factor; Neg, negative; PDGF, platelet-derived growth factor; Pos, positive; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinases; TNF, tumor necrosis factor; uPAR, urokinase plasminogen activator receptor.

Evidence for Direct Involvement of uPA and uPAR in Stem Cell Migration
To show a direct cause-and-effect relationship between uPAR expression on the tumor cells and uPA in the tumor-conditioned media, we focused on the PC-3 prostate cancer cell line and used function-inhibiting anti-uPAR and anti-uPA antibodies and siRNA-mediated gene silencing. To show a direct effect of uPA on stem cell migration, we used purified human recombinant uPA as a chemoattractant for hfMSCs and HB1.F3 stem cells (Fig. 5A). Migration of both mesenchymal and neural stem cells was induced by addition of uPA to serum-free medium, and we observed highest migration at 0.25 ng/ml of uPA. At all concentrations of uPA tested (0.1–100 ng/ml), the NSCs showed higher migration than MSCs, which is consistent with data from migration assays using tumor cell-conditioned media.

View larger version (36K): [in this window] [in a new window]   Figure 5. Induction of stem cell migration by recombinant uPA and inhibition by uPA depletion and uPAR function-inhibiting antibodies or siRNA knockdown. (A): Migration of HB1.F3 cells and hfMSCs to human recombinant uPA protein. (B): Inhibition of HB1.F3 neuronal stem cell migration to tumor-derived conditioned media after preincubation with anti-uPAR antibodies for 2 hours prior to cell migration assay. (C): Migration of HB1.F3 cells to conditioned media derived from control PC-3 cells or siRNA knockdown of uPAR. Inset, uPAR expression in PC-3 cell was confirmed by flow cytometry analysis after 48 hours of siRNA treatment. (D): Migration of HB1.F3 cells to PC-3 cell-derived conditioned media was inhibited by using anti-PA antibodies bound to protein A-Sepharose beads (isotype-matched IgG was used as control). Five percent BSA and 10% FBS were used as negative and positive controls for migration assays, respectively. Data are expressed as mean + SD of triplicate measurements. Three independent experiments were performed. Abbreviations: BSA, bovine serum albumin; FBS, fetal bovine serum; hfMSC, fetal human bone marrow-derived primary mesenchymal stem cell; siRNA, small interfering RNA; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor.

Since our data showed that expression of uPAR on tumor cells correlated with stem cell tropism (Fig. 2A), we reasoned that siRNA-mediated inhibition of uPAR expression on PC-3 cancer cells would result in inhibition of HB1.F3 neural stem cell migration. As shown in Figure 5C, conditioned media derived from PC-3-uPAR-siRNA cells, compared with conditioned media from PC-3 control cells, displayed inhibition of attraction of HB1.F3 cells by 70%–90%. Furthermore, HB1.F3 cell migration to PC-3-conditioned medium was inhibited by depletion of uPA from PC-3-conditioned medium using anti-PA antibodies bound to protein A-Sepharose beads (>90% of inhibition; Fig. 5D).

Transduction of Tumor Cells with uPA and uPAR Genes Induces Stem Cell Attraction In Vitro
To further investigate the role of uPA and uPAR expression in stem cell tropism to tumors, we transduced NB1691 neuroblastoma cells (a low uPA and uPAR-expressing line) with pBABE.uPA and pBABE.uPAR retroviral vectors. Overexpression of uPA and uPAR was confirmed by quantitative real-time RT-PCR analysis (data not shown). We used in vitro cell migration assays to test whether NSCs (HB1.F3 and HB1.F5) and MSCs (hfMSCs) would migrate toward conditioned media derived from the transduced NB1691 cells (Fig. 6A, 6B). Retroviral vector-mediated overexpression of uPAR alone on NB1691 neuroblastoma cells leads to threefold and twofold greater HB1.F3 and hfMSC migration, respectively. uPA overexpression in NB1691 cells, however, caused 15-fold and 6-fold greater NSC and MSC migration, respectively, compared with the NB1691 wild-type cells. Coexpression of uPA and uPAR in NB1691 cells resulted in stem cell migration comparable to that toward PC-3 conditioned media, which induced the greatest migration of stem cells in previous experiments (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Figure 6. NSC (HB1.F3) and hfMSC migration toward NB1691 neuroblastoma cells overexpressing uPA and uPAR using pantropic retroviral expression system. Transgene expression was confirmed using real-time polymerase chain reaction (data not shown). Migration of NSCs (A) and mesenchymal stem cells (B) to NB1691.wt, NB1691.uPA, and NB1691.uPAR-derived conditioned media. FBS (10%) and PC-3 conditioned media were used as positive control, whereas BSA (2%) served as negative control. Error bars indicate SD of triplicate measurements from two independent cell migration assays. Abbreviations: BSA, bovine serum albumin; FBS, fetal bovine serum; hfMSC, fetal human bone marrow-derived primary mesenchymal stem cell; NSC, neural stem cell; uPA, urokinase plasminogen activator; uPAR, urokinase plasminogen activator receptor.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

Here we report that both NSCs and MSCs show significantly greater migration toward cancer cells that express high levels of uPA and uPAR, compared with tumor cells with low expression of uPA and uPAR. Activation of the uPA/uPAR system on cancer cells caused the release of uPA and suPAR into the conditioned media. Furthermore, expression of uPA and uPAR on cancer cells was associated with secretion of several cytokines, including HGF, IL-6, IL-8, and MCP-1 (CCL2). Comparative analysis of cytokine profiles of conditioned media showed that IL-6 and IL-8 were highly expressed in MDA-MB-231 cells, compared with MCF-7 breast cancer cell line. High levels of IL-8 and TIMP-2 were secreted into conditioned media from H1915 cells, compared with H1415 lung cancer cells, whereas conditioned media from SK-N-AS neuroblastoma cells had elevated MCP-1 and HGF. The PC-3 prostate cancer cell line, which displayed the highest uPAR expression and chemoattraction of stem cells, had elevated levels of all the aforementioned cytokines secreted into the conditioned media. These data suggest that NSCs and MSCs can use multiple cytokines for tropism to tumors but that a common feature of tumors that attract stem cells is that they all express uPA and uPAR. We observed induction of stem cell migration to human recombinant uPA, whereas significant inhibition of stem cell migration was observed after blocking uPAR on the stem cells and tumors or after depletion of uPA from tumor cell-conditioned media. Overexpression of uPA and uPAR alone or in combination resulted in increased migration of NSCs and MSCs to conditioned media from NB1691 neuroblastoma cells. The chemoattracting activity of uPA may occur directly and/or indirectly by causing the induction of other cytokines, such as IL-6, IL-8, and MCP-1. There is evidence that cancer cell migration and invasion, which are modulated by uPAR, involve HGF, insulin-like growth factor, matrix metalloproteinase (MMPs), IL-6, IL-8, and MCP-1, which suggests that cancer cells and stem cells may use similar signaling pathways during migration [34]. Earlier reports suggest that nuclear translocation of β-catenin leads to simultaneous activation of target genes, such as uPAR and IL-8 [35]. Furthermore, activation of the IL-6 and IL-8 genes leads to increased expression of uPAR, MMP2, and MMP9 in solid tumors [36]. TIMPs are endogenous inhibitors of MMPs that have recently been identified as signaling molecules, and they act via the MEK1-Erk signaling network, similarly to uPA and uPAR [37]. In summary, our data suggest that uPA and uPAR underlie a novel mechanism of stem cell tropism to invasive solid tumors. Identification and characterization of cancer-associated cytokines, specific for various phenotypes and tumor origins, will be important in the development of effective stem cell-based targeted cancer therapies.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We thank Dr. Kristine A. Justus and Dr. Keely Walker for expert editing of the manuscript. We also thank Dr. Chu-Chih Shih for kindly providing mesenchymal stem cells. This work was supported by National Institutes of Health/National Cancer Institute (CA113446), The Stop Cancer Foundation, Joseph Drown Foundation, The Rosalinde and Arthur Gilbert Foundation, Neidorf Family Foundation, H.L. Snyder Foundation, and Ziman Family Foundation.

	FOOTNOTES

 
Author contributions: M.G.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; J.N.: data analysis and interpretation, collection and assembly of data, manuscript writing, final approval of manuscript; R.T.F.: collection and assembly of data, data analysis and interpretation; S.E.K., M.G., M.E., D.Z.: collection and assembly of data; A.G.: conception and design; M.Z.M.: collection of data, data analysis; C.A.G.: data analysis and interpretation; S.U.K.: provision of study material or patients; K.S.A.: financial support, administrative support, provision of study material or patients, data analysis and interpretation, manuscript writing, final approval of manuscript.

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