1 Department of Oncology, 2 Department of Clinical Neurosciences, 3 Department of Biochemistry and Molecular Biology and 4 Department of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada
* To whom correspondence should be addressed at: Department of Oncology and Department of Clinical Neurosciences, University of Calgary, 3330 Hospital Drive, Calgary, Alberta T2N 4N1, Canada. Tel: +403 220 3544; Fax: +403 283 8731; E-mail: vyong{at}ucalgary.ca
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Abstract |
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Abbreviations: CNS, central nervous system; ELISA, enzyme linked immunosorbent assay; ECM, extracellular matrix; GBM, glioblastoma multiforme; H&E, hematoxylin and eosin; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; MMP, matrix metalloproteinase
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Introduction |
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Chemokines are a family of molecules that were described originally to regulate the chemotaxis of leukocytes into tissues. It is now clear that chemokines have other roles, including promoting mitosis, survival and angiogenesis (reviewed in Refs 7 and 8). In view of these characteristics, it is not surprising that chemokines are now implicated in the pathogenesis of a variety of tumors (811). Several chemokines have been described on glioma cells and these include macrophage chemoattractant protein-1 (CCL2) (12,13), interleukin-8 (CXCL8) (14), RANTES (CCL5) (15) and stromal cell-derived factor-1 (CXCL12) (16,17). Chemokine receptors are also expressed on glioma cells. We described the predominance of CXCR4 in a majority of glioma lines and that its ligand, CXCL12, is a survival and chemotactic factor for glioma cells in vitro (18). It is likely that the roles of chemokines in glioma pathophysiology will expand.
The chemokine GRO- (growth related oncogene-
, CXCL1) is a growth factor as well as a chemoattractant molecule. It is implicated as an oncogenic factor in several tumor types, including prostate and melanoma cancers (19,20). Of possible relevance to malignant gliomas is the observation that GRO-
synergizes with platelet-derived growth factor to increase the proliferation of oligodendrocyte precursors during development (2123). Furthermore, resected specimens of oligodendrogliomas were shown to express GRO-
protein (24).
In this study, we have investigated whether the GRO- protein can regulate the tumorigenic potential of glioma cells, and through what mechanisms. Our results implicate GRO-
as an oncogenic factor in glioma biology.
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Materials and methods |
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GRO- cloning and plasmid construction
To clone the human GRO- gene, cDNA was derived from the human astrocytoma cell line, U87, using reverse transcription. Four µg of total RNA from U87 cells was incubated with random primers (Roche, Indianapolis, IN), Superscript II reverse transcriptase (Invitrogen, Burlington, Ontario), and RT buffer at 37°C for 2 h. The coding sequence of the GRO-
gene was obtained by PCR amplification from U87 cDNA using the following oligonucleotides: sense 5'-CAC AGA GCC CGG GCC GCA GGC ACC-3', antisense 5'-ATA AGG GCA GGG CCT CCT TCA GG-3'. The resulting 460 bp PCR product was subcloned into the PCR cloning vector pGEM-T easy (Promega, Madison, WI) and sequenced to confirm accuracy. The GRO-
cDNA was then cloned into the Not1 site of the expression vector pcDNA3.1+ and named pcDNA3.1-GRO-
.
Enhanced green fluorescent protein (EGFP) coding sequence was obtained by digesting pEGFP (Clonetech, Palo Alto, CA) with XbaI. Gel-purified fragment was cloned into the XbaI site of pcDNA3.1(+) to yield plasmid pcDNA3.1-EGFP. The clones were screened to ensure that EGFP was inserted in the sense orientation.
RTPCR
For total RNA isolation, cells at 80% confluency in 100-mm dishes were lysed in 1 ml TRIzol (Invitrogen). RNA was isolated following the manufacturer's instruction and was reconstituted in 25 µl RNaseOUT (Invitrogen) water. Trace DNA was removed by DNase digestion at 37°C for 45 min. Reverse transcription was performed by incubation of 4 µg of total RNA with random primers (Roche), Superscript II (Invitrogen) and RT buffer at 37°C for 2 h. An aliquot of 1/10 of the RT product was applied for PCR using Taq polymerase (Invitrogen). The GRO- primers were, forward: 5'-AGG GAA TTC ACC CCA AGA AC-3', and backward: 5'-TGG ATT TGT CAC TGT TCA GCA-3'. The GAPDH primers were forward: 5'-CCA TGG AGA AGG CTG GGG-3' and backward: 5'-CAA AGT TGT CAT GGA TGA CC-3'. The PCR products were amplified for 35 cycles at 94°C for 45 s, 55°C for 45 s and 72°C for 45 s, and were visualized by 1% agarose gel electrophoresis.
Tissue culture and stable transfection
The U251 human glioma cell line and other lines displayed in Figure 6 have been used in previous studies (18,25); U251 originates from a glioblastoma (WHO Grade IV) specimen (26). One µg of pcDNA3.1-GRO- and 1µg of pcDNA3.1-EGFP were co-transfected with Lipofectamine according to the manufacturer's recommended protocol (Invitrogen). Selected clones of G418 (Calbiochem Novabiochem, San Diego, CA) were tested for GRO-
production using enzyme linked immunosorbent assay (ELISA) (see below) and EGFP expression by immunofluorescence microscopy of live cultures. Clones expressing both GRO-
and EGFP were further selected and kept at all times in the presence of 500 µg/ml G418.
As control, 1 µg of pcDNA3.1-EGFP was transfected into U251 cells. Fluorescent clones were pooled and used as vector control line in all the experiments of this study.
GRO- ELISA
Fifty thousand glioma cells were seeded into each well of a 24-well plate. The amount of GRO- secreted into the medium after 48 h of culture was analyzed using 100 µl of cell-conditioned medium. GRO-
ELISA was analyzed as described by the manufacturer (R&D Systems).
Cell growth curve
Ten thousand cells were plated into each well of a 24-well plate in 1 ml of medium. For the next 4 days at 24 h intervals, cells were treated with 0.25% trypsin in order to detach cells into the buffer. The entire content of each well was then transferred to vials containing 9.5 ml of phosphate buffered saline (PBS) and counted by a Z2 Coulter Counter (3 µm gate). The resultant values obtained represented the total number of cells per well. Cell numbers in log scale were plotted against the time points, giving rise to the growth curve of cells.
Invasion assay
BioCoat GFR Matrigel Invasion Chambers with 8 µm pore size (BD Biosciences, Mississauga, Ontario) was employed to assess invasive properties of transfectants as per protocols provided by the manufacturer. Briefly, 1 x 104 cells suspended in serum-free medium were seeded into the insert of each chamber. Medium containing 10% fetal calf serum was added to the bottom well so that the chemoattractant force would direct the cells to invade across the Matrigel coated membrane. The entire chamber set-up was incubated at 37°C for 48 h. Glioma cells that invaded across the membrane tended to remain adherent to the underside of the barrier. Cells were fixed in 95% ethanol/5% acetic acid for 15 min followed by hematoxylin staining for 15 min. A cotton-tipped swab was applied to remove the cells in the upper compartment that did not invade across. The membrane was then cut off and the number of invaded cells on the underside was counted either across the entire membrane (Figure 5) or in six random fields (Figure 6).
Migration assay
Two different methods were used. In the first (scratch assay), 100 000 cells were plated onto each of 12 mm diameter glass cover slips in order to rapidly obtain a very confluent monolayer of cells. One day after, a rubber policeman was used to scrape cells off from half the cover slip. The initial confluent monolayer allowed a reliable evaluation of where the scratch line (point of origin of migration) was (27). The distance that remaining cells migrated across the scratch line was then assessed 48 h after, following the fixation of cells in 95% ethanol/5% acetic acid (v/v) and staining with hematoxylin for 15 min. Cells were then photographed using a 20x objective, imported via a CCD camera into Image-pro software, and the distance that cells have migrated was then measured on monitor in mm. In some experiments, hepatocyte growth factor (HGF) was added at a final concentration of 100 ng/ml as a positive control to stimulate migration of cells.
The second method involved the plating of cells onto the 8 µm invasion chambers described earlier, except that there was no extracellular matrix (ECM) protein coating that would necessitate the production of proteases by glioma cells to mimic an invasive process; there was also no chemokine gradient. Rather, the absence of protein coating and chemokine allowed the random migration of glioma cells in all directions including to the underneath surface of the membrane; the number of cells migrated was then counted in six randomly selected fields.
Assessment of ß1-integrin expression by flow cytometry
Cells at 80% confluency were scraped off 100-mm dishes using a rubber policeman, and 106 cells were stained with a monoclonal anti-ß1-integrin (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 45 min. Cells were washed, then stained with secondary antibody, which was goat anti-mouse IgG conjugated to PE (Caltag Laboratories, Burlingame, CA), for another 45 min at 4°C. After washing and fixation, the cells were analyzed by flow cytometry.
Adhesion assay
This was performed as described previously (25). For this study, 96-well plates were employed and 2.5 x 104 cells were seeded per well. The amount of ECM proteins used was 10 µg/ml.
Analyses of matrix metalloproteinase-2 level and activity
A gelatin zymography protocol was used (6,28) to analyze the amount of pro-matrix metalloproteinase (MMP)-2 produced by cells. Cells (2.5 x 105) seeded into 24-well plate were fed with 0.5 ml of serum-free medium. Cell-conditioned medium was collected 48 h after and was analyzed for MMP-2 content. Zymography measures the ability of proteases to degrade gelatin, and is dependent on the amount of protease present, rather than on its intrinsic activity. Gelatin zymography commonly detects the pro-form of MMP-2 and MMP-9, but not the active forms. Thus, to measure the amount of active MMP-2 that is present in the cell-conditioned medium, a MMP-2 fluorogenic substrate, MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH2 (Calbiochem) was utilized (6). An aliquot of 10 µl of 48 h cell-conditioned medium was added to individual wells of a 96-well culture plate. An aliquot of 50 µl of 50 µM stock MMP-2 substrate was then added to the wells. Fluorescence was monitored every 10 min for 2 h in a SpectraMAX (Molecular Devices, Sunnyvale, CA) with excitation wavelength of 325 nm and emission wavelength of 393 nm, and expressed as relative fluorescence units.
DNA microarray
GRO- clones 5 and 54, and vector control line, were seeded at 2 million cells into 100-mm dishes and incubated at 37°C with 5% CO2 overnight. RNA was extracted from the cells with TRIzol. Total RNA was treated with DNAse to remove genomic DNA (MessageCleanTM). Labeled cDNA was prepared from 10 µg of total RNA using the FairplayTM Microarray Labeling Kit (Stratagene, Mississauga, Ontario) according to the manufacturer's recommendations. The cDNA pellet was resuspended in 5 µl of 2x coupling buffer, incubated at 37°C for 10 min, and then combined with 5 µl of monoreactive dye (Amersham Biosciences, Piscataway, NJ) dissolved in DMSO, and incubated for 30 min at room temperature in the dark. The dye-coupled cDNA was purified through a spin column, and eluted with 10 mM TrisHCl, pH 8.5, and ethanol precipitated. Hybridization mix was prepared by combining 90 µl DIG EasyHybTM (Roche), 5 µl yeast tRNA (10 mg/ml), and 5 µl fish sperm DNA (10 mg/ml). The appropriate labeled cDNA pellets were dissolved each in turn in a total of 5 µl TE, combined with 65 µl of the prepared hybridization mix, and heated in a 65°C water bath for 2 min, and cooled to room temperature. Human arrays with 14 000 70mer oligos spotted in duplicate were obtained from the Southern Alberta Microarray Facility (University of Calgary). The slides were incubated at 37°C overnight in a hybridization oven. The following day, the slides were taken out of the oven, and washed at room temperature with constant agitation in the following washes: 2 x SSC, 0.2% SDS for 5 min , 0.2 x SSC for 5 min, and 0.05 x SSC for 5 min. After the final wash, the slides were placed in a slide rack and spun dry at 500 r.p.m. for 5 min in a benchtop centrifuge to remove all excess liquid. The slides were then scanned in a PerkinElmer ScanArray 5000TM using the Green HeNe 543.5 nm laser for excitation of Cy3 and the Red HeNe 632.8 nm laser for excitation of Cy5. The scans were saved as image files in TIFF format, and imported into the QuantArrayTM version 3.0 (PerkinElmer, Boston, MA) microarray analysis software, for spot identification and quantification, and background estimation. The quantification and image files were then loaded into Gene Traffic DuoTM (Iobion, La Jolla, CA) for microarray data management and analysis. The data were filtered to flag spots with intensities <100 U, or less than twice the average background. The data were normalized according to the Lowess method (29) resident in the Gene Traffic software. We focused on genes that were altered in common between the two GRO-
clones when compared with the vector control cells.
Tumor cells implantation, and assessment of survival and tumor size
GRO- clones and vector control line were harvested with trypsin, washed with PBS once and resuspended in PBS at a concentration of 2.5 x 107/ml. For each anesthetized nude mouse, 4 µl of cells (1 x 105) was injected into the right striatum. Animals were sutured and returned to their cages and allowed ad libitum access to food and water. Animals were weighed every other day and observed for symptoms of neurological deficit. To obtain data for the KaplanMeier survival curve, mice found dead in their cages were recorded; in most cases, however, in accordance with humane practice, mice that were moribund (negligible limb movement, and loss of body weight exceeding 20%) were considered dead. All protocols are approved by the Animal Care Committee at the University of Calgary in accordance with research guidelines from the Canadian Council for Animal Care.
In another series of experiments, in order to address tumor growth in the brain prior to the appearance of symptoms, four mice each were implanted with vector or GRO- expressing cells as described above and killed 14 days after. The whole brain was removed, cut into blocks, fixed in formalin and embedded in paraffin. Sections of 6 µm were taken every 200 µm apart, through the entire brain. Sections were heated for 1 h at 60°C, deparaffinized in xylene, and rehydrated successively through a graded series of alcohol and PBS. Sections were then stained with hematoxylin and eosin (H&E), dehydrated through a graded series of alcohol and xylene, and mounted with Acrotol. All sections were analyzed for the presence of tumor, and thus, the entire rostral caudal extent of the brain occupied by tumor could be determined.
Some sections were also stained with a rabbit polyclonal antibody to GFP, followed by immunoperoxidase and hematoxylin counter stain. These analyses confirmed the correspondence of tumors detected by routine H&E and GFP immunoreactivity.
Statistics
When two groups were compared, the unpaired Student's t-test was applied. When multiple groups were evaluated, the one way ANOVA test with post-hoc TukeyKrammer multiple comparisons was used. Statistical significance was set at P < 0.05.
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Results |
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The capacity of cells to be more motile in the scratch assay was indicative of the ability of intact cells to interact with the ECM deposited by the cells that were previously seeded and then scratched off the cover slip. To confirm this, we assessed the ability of transfectants to adhere on various ECM substrates. Figure 3C demonstrates that the GRO- expressing clones were more adherent onto fibronectin, laminin and vitronectin substrates.
Integrins are receptors on cells that interact with ECM proteins. The results of increased adhesiveness of GRO- transfectants suggested the upregulation of integrins on glioma cells that overexpressed GRO-
. This was confirmed at the level of flow cytometry where GRO-
transfectants displayed higher levels of the ß1-integrin, which is a receptor subunit for the ECM proteins examined in this study (31).
Altogether, these results demonstrate that while the GRO- expressing glioma cells did not have an increased growth rate in culture, they were more motile and had greater capacity to interact with ECM proteins. No obvious differences between the GRO-
5 and 54 clones were found in these assays.
Increased MMP-2 production and invasiveness of GRO- transfectants in vitro
We determined the expression of MMP-2 in the GRO- transfectants, as MMP-2 is implicated in the invasiveness of glioma cells in vitro and in vivo (6,28,3234). MMP-2 is a secreted protease, enabling cell-conditioned media to be used for analyses. Gelatin zymography demonstrated the increased level of pro-MMP-2 in the GRO-
clones compared with vector control cells; the higher GRO-
expressing clone, 54, had a correspondingly greater production of pro-MMP-2 than the lower GRO-
expressing clone 5 (Figure 4A). This was not the case for pro-MMP-9, which, indeed, may be decreased by the GRO-
expression.
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The increased MMP-2 production and activity suggests a more invasive phenotype of GRO- transfectants. To test this in vitro, the ability of glioma cells to invade across a reconstituted basement membrane, Matrigel, was evaluated. Figure 5 shows that over a 48 h invasion period, the GRO-
transfectants were more invasive than vector control cells.
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Microarray analyses of GRO- transfectants
Vector control cells and the GRO- 5 and 54 clones were subjected to microarray analyses to investigate whether other proteins were also affected in common in the GRO-
transfectants. Of 14 000 genes analyzed, only 4 genes in common between the two GRO
transfecants were upregulated compared with the vector control cells: vimentin, SPARC, phosphoglycerate kinase 1 and glyceraldehyde-3-phosphate dehydrogenase (Table I). It should be noted that GRO-
and MMP-2 were not on the array. The identification of SPARC is of interest as this protein is upregulated in diffuse low grade astrocytoma (WHO Grade II) compared with normal brain specimens (35), where its function may be to mediate the invasiveness of glioma cells (36,37).
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Discussion |
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The chemokine family has received increased attention as multi-functional proteins that regulate many cellular phenotypes in addition to their classical roles as chemotactic molecules. In the context of tumor biology, specific chemokines are angiogenic or angiostatic (42,43), and they regulate survival, cell cycle progression, growth and cellcell interaction (7,8,44). The preferential metastatic spread of certain cancers to particular organs appears to be controlled by chemokine receptors on tumor cells homing to signals generated by specific chemokines expressed by the target tissue (9). Not surprisingly, chemokines and their receptor systems are being actively pursued as novel cancer therapeutics (45).
As noted earlier, several chemokines have now been described on glioma cells in situ and in vitro. The function of these proteins in glioma biology remains uncertain but they could account for the increased malignancy of glioma cells. For example, hypoxic/anoxic insults to glioma cells in vitro induced an increase in IL-8 mRNA, which is thought to be important in the generation of new vessels for increased oxygenation and blood supply (14). MCP-1-transfected rat glioma cells were massively infiltrated by microglia when implanted in vivo, promoting tumor growth (46). We reported recently that the widespread expression of the CXCR4 chemokine receptor on glioma cells may mediate not only chemotaxis, but also survival functions (18). Others have proposed a role for CXCR4 and its ligand, CXCL12, in angiogenesis in glioma specimens (16,17).
In this study, we have addressed the role of GRO- in modulating the phenotype of gliomas. GRO-
is of particular interest since it is implicated in modulating the proliferation of oligodendrocyte progenitor cells in development (2123). We found that the expression of GRO-
in the U251 line confers onto these cells various characteristics compatible with increased tumorigenicity, such as increased motility and invasiveness in vitro, and enhanced tumor spread in the brain in vivo. These changes correspond with increased MMP-2 expression and activity, and elevated ß1-integrin level, mechanisms that are implicated in regulating the aggressiveness of many types of tumor cells, including gliomas (28,3234,47).
In addition to MMP-2 and integrins, microarray analyses revealed other molecules that were regulated by GRO-. The elevation of the glycolytic enzymes, phosphoglycerate kinase 1 and glyceraldehyde-3-phosphate dehydrogenase, is reflective of a more metabolically active state, while the increase of SPARC has direct relevance to invasiveness. SPARC is a 43 kDa secreted matricellular glycoprotein that interacts with ECM components to regulate adhesion and de-adhesion events, among others, that are conducive for cell motility; high levels of SPARC are usually associated with metastatic tumors (reviewed in Ref. 48). SPARC is expressed in human glioma specimens in situ (35,36,49), and its overexpression in glioma cells confers altered rates of migration in vitro (50) and in tumor models in vivo (37). Thus, the findings of this manuscript reveal at least one mechanism that accounts for the upregulation of SPARC in glioma cells: GRO-
expression.
It is unknown if the elevated vimentin expression in the GRO- clones has functional significance to the glioma cells with respect to growth or invasive properties. Vimentin is an intermediate filament protein and its co-expression with nestin has been suggested to serve as a marker for an astrocytoma cell type with enhanced motility and invasive potential (51). When another intermediate filament protein, GFAP, was underexpressed in invasive astrocytoma cells through antisense transfection, this resulted in decreased invasiveness of cells (52,53). Thus, it is possible that the vimentin upregulation in the GRO-
clones helps account for the increased invasiveness of glioma cells.
The microarray analyses have also shown the downregulation of insulin-like growth factor (IGF) binding protein 7 (Table I). The significance of this finding is speculative but its decreased level could make more IGF bioavailable; elevated IGF-1 has been associated with the increased invasiveness of glioma cells (54). In contrast, however, it has been noted that IGF binding protein 2 enhances glioma invasiveness (55).
Although our results reveal some mechanisms whereby GRO- expression confers increased tumorigenicity to glioma, e.g. through MMP-2 and SPARC levels, it is unknown how precisely these mechanisms were achieved. GRO-
is exported out into the extracellular fluid by glioma cells, since high levels are detected by ELISA analyses of the conditioned medium. However, we have noted that the majority of glioma lines, including U251, do not express the principal receptor for GRO-
, CXCR2 (18), suggesting that autocrine growth regulation by GRO-
is unlikely to be important in this study. Furthermore, U251 cells (wild-type, vector- or GRO-
-transfected) did not contain detectable levels of CXCR2 on their cell surface as addressed by flow cytometry (data not shown). Finally, exogenous GRO-
added onto U251 wild-type or vector-transfected cells did not increase the motility or invasiveness of these cells (data not shown). We are left with the possibility that GRO-
overexpressed in U251 glioma cells mainly acts through intracellular (intercrine) means to regulate the cell phenotype. Such a possibility remains to be investigated in future experiments, but we note that some molecules, such as fibroblast growth factor-2, have been shown to alter cell characteristics by a mechanism that involves intracellular and nuclear mechanisms, rather than the activation of extracellular cell surface receptors following its secretion (5658).
Many tumor cell types exploit their environment for their growth, survival and invasive capacity (59). It is also clear that inflammatory molecules have a role in modulating the growth of tumor cells in several cancers (60). Thus, besides an intracellular mechanism of action, it is possible that, in vivo, the secretion of GRO- from the U251 clones into the intracerebral mouse environment activates CXCR2 on host cells which then provide factors conducive to the growth of the U251 GRO-
clones.
How widespread is GRO- expression in brain tumors in situ? In our study, both glioma specimens contained GRO-
immunoreactive cells. In the only other publication of GRO-
expression in glioma specimens that we are aware of, 7 of 7 oligodendrogliomas and 6 of 10 glioblastomas (WHO Grade IV) contained GRO-
immunoreactive tumor cells; 0 of 4 diffuse astrocytomas (WHO Grades II and III) were positive (24). Thus, GRO-
expression appears to be common in oligodendrogliomas and can be found in a significant number of astrocytomas, particularly the higher grade ones. It would be interesting to ascertain whether the MMP-2, SPARC and integrins that are commonly overexpressed in gliomas is a result of the GRO-
upregulation.
In summary, we have found that a proliferative factor for oligodendrocyte precursor cells, GRO-, confers onto glioma cells several properties associated with the malignant phenotype, indicating that the dysregulation of glia proliferative factors contributes to tumorigenesis. We suggest that GRO-
expression is a negative prognostic factor in gliomas, and that the targeting of this chemokine may constitute a useful approach to curb the high morbidity that is associated with malignant gliomas.
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Acknowledgments |
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Conflict of Interest Statement: None declared.
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References |
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