Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218
Submitted 17 October 2003 ; accepted in final form 30 March 2004
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ABSTRACT |
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P-selectin; IIb
3-integrins; shear stress
The physiological relevance of secondary tethering in leukocyte adhesion is under active investigation. These interactions were observed originally in vitro during leukocyte-only perfusions over adhesive substrates (2, 36). However, both in vitro and in vivo studies in which red blood cells (RBCs) are present in flow in addition to leukocytes offer conflicting results (12, 23, 30, 33, 34). An in vitro study using diluted and undiluted whole blood specimens rather than isolated leukocyte suspensions demonstrated that secondary tethering plays a major role in enhancing leukocyte adhesion to purified P-selectin (34). Along these lines, an in vivo study revealed that L-selectin-mediated secondary tethering contributes significantly to leukocyte rolling flux in arterial vessels and large venules (>45-µm diameter) but not in smaller venules (12), whereas another investigation observed secondary tethers in venules as small as 10 µm in diameter (33). In contrast, previous work by Mitchell et al. (30) involving whole blood perfusions questioned the occurrence of secondary neutrophil tethering to E-selectin-coated substrates. Yet another study showed that, although leukocyte-leukocyte tethers can occur in vivo, these interactions are not the major cause of leukocyte clustering observed in venules with diameter of 28108 µm (23). Despite the debate surrounding leukocytes, no investigation has yet addressed whether tumor cells are capable of secondary tethering.
It was shown previously that platelet microparticles are capable of mediating secondary tethers by bridging free-flowing L-selectin-negative HL60 myeloid cells and L-selectin-antibody-blocked neutrophils with immobilized cells of the same type (15). This interaction occurs via microparticle P-selectin binding to HL60 or neutrophil PSGL-1 (15). However, the potential involvement of other cell adhesion molecules such as IIb
3-integrin was not examined. Moreover, the degree of leukocyte coating by platelet microparticles necessary to support leukocyte bridging was not reported, and whether intact platelets could similarly support secondary tethers remains to be determined. Most importantly, the physiological relevance of the platelet-mediated secondary tethering mechanism in the presence of RBCs has yet to be established.
Other studies have instead implicated leukocyte-adherent platelets capable of enhancing adhesion by forming a bridge between the leukocyte and the endothelial cell. For instance, thrombin receptor-activating peptide (TRAP)-stimulated, but not nonstimulated, platelets bound to leukocytes in vivo were able to form an adhesive bridge to facilitate leukocyte attachment to high endothelial venules (HEVs) of peripheral lymph nodes when L-selectin function was inhibited (11). These interactions occurred through platelet P-selectin and P-selectin counterreceptor(s) on the surface of HEVs (11). In addition, a previous study using the THP-1 monocytoid cell line demonstrated that TRAP-activated platelets can assist in the deposition of THP-1 cells to PMA-stimulated endothelium at high shear rates at which these cells alone would not otherwise tether (400 s1) (35). Neither of these investigations identified a role for platelets in mediating secondary tethering whereby a platelet transiently bridges an endothelium-adherent with a free-flowing leukocyte before the latter attaches to the endothelial surface. Similarly, platelet-tumor cell perfusions over components of the extracellular matrix (ECM) indicate an important role for platelets in promotion of tumor cell adhesion by bridging tumor cells to the ECM, yet secondary tethering was not evaluated (9, 13, 14).
Accumulating evidence provides strong support for the concept that platelets facilitate the hematogenous dissemination of tumor cells, the most convincing of which is the inhibition of metastasis by experimental platelet depletion and the reconstitution of metastatic potential after platelet repletion (16, 19). It is believed that platelets provide a protective shield against cytotoxic immune cells (32) and facilitate tumor cell extravasation by potentiating tumor cell adhesive interactions with ECM proteins of the vessel wall (9, 13). Morphological observations of tumor cells arrested in capillaries (24) and in lung microvasculature (4) revealed the close association of tumor cells with activated platelets. Earlier work in our laboratory demonstrated that metastatic LS174T colon adenocarcinoma cells interact extensively with immobilized platelets (27) as well as activated platelets in bulk suspensions (25) under dynamic shear conditions. However, the effects of platelets on LS174T cell adhesion to vascular endothelium in the presence and absence of RBCs have yet to be evaluated. We therefore wished to examine in a systematic manner whether platelets form a bridge between a free-flowing tumor cell (lacking L-selectin) and an endothelium-adherent tumor cell to mediate secondary tethering and/or bridge adhesion between a free-flowing tumor cell and the endothelial cell monolayer. These possibilities warrant investigation, because elucidating the role of platelets in metastasis may provide insights into the development of novel therapeutic strategies to combat the spread of cancer.
To examine the role of platelets in adhesion of colon carcinoma to vascular endothelium, thrombin-GPRP-treated washed platelets were simultaneously perfused with metastatic LS174T colon adenocarcinoma cells over 4-h TNF--stimulated human umbilical vein endothelial cells (HUVECs) in a parallel plate flow chamber system. The effects exerted by varied platelet concentration, wall shear stress and rate, and addition of RBCs on primary and secondary tethering of tumor cells were systematically investigated. Efforts were then focused on identifying the adhesion molecules involved in the platelet-induced enhancement of tumor cell adhesion. Determinations made with LS174T carcinoma cells were also compared with results obtained from experiments with monocytoid THP-1 cells, also lacking L-selectin, to ensure the generality of the results.
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MATERIALS AND METHODS |
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Monoclonal antibodies and adhesion antagonists.
All antibodies were murine anti-human IgG1 unless otherwise noted. The blocking monoclonal antibodies (MAbs) AK4 (anti-P-selectin), Dreg 56 (anti-L-selectin), and HIP1 [anti-glycoprotein Ib (GPIb)] were purchased from Pharmingen (San Diego, CA). Blocking anti-ICAM-1 MAb (BBIG-I1) was obtained from R&D Systems (Minneapolis, MN). Blocking anti-L-selectin MAb LAM1116 was kindly donated by Dr. T. F. Tedder (Duke University Medical Center, Durham, NC; Ref. 10). Blocking anti-IIb
3 Fab fragment c7E3 was from Centocor (Malvern, PA). A FluoroTag FITC conjugation kit (used to label c7E3 with FITC) and an isotype-matched IgG1 MAb were purchased from Sigma (St. Louis, MO). Nonpeptide small-molecule antagonists XV454 (
IIb
3 antagonist) and XT199 (
v
3 antagonist) were generously provided by Dr. S. A. Mousa (Albany College of Pharmacy, Albany, NY; Ref. 1).
Cell culture.
LS174T human colon adenocarcinoma cells and THP-1 myeloid cells were obtained from the American Type Culture Collection and cultured in the recommended medium. LS174T cells were detached from culture flasks by mild trypsinization (0.25% trypsin-EDTA for 2 min at 37°C; Life Technologies) and subsequently incubated at 37°C for 2.5 h to regenerate surface glycoproteins (6, 17, 27). Suspension-cultured THP-1 cells were washed, resuspended at 107 cells/ml in serum-free medium containing 0.1% bovine serum albumin (Sigma), and stored at 4°C for no longer than 5 h before use in flow-based adhesion assays. Viability assessed by Trypan blue exclusion was routinely 97%.
HUVECs were harvested by collagenase digestion and cultured to confluence in 1% gelatin-coated tissue culture flasks as described previously (6, 22). Cells were then passaged into gelatin-coated 35-mm tissue culture dishes. Before use in adhesion assays, first-passage HUVECs were stimulated for 4 h with 5 ng/ml TNF- (R&D Systems) to obtain maximal levels of E-selectin expression.
Preparation of washed platelets and washed RBCs. Washed platelets were prepared as described previously (26). Briefly, platelet-rich plasma (PRP) was prepared by centrifugation (160 g, 15 min) of 0.38% (wt/vol) sodium citrate-anticoagulated human blood from healthy volunteers. PRP specimens were then subjected to centrifugation (1,100 g, 15 min) in the presence of 2 µM PGE1. Isolated platelets were resuspended in HEPES-Tyrode buffer containing 5 mM EGTA and 2 µM PGE1, washed once via centrifugation (1,100 g, 10 min), and resuspended at 2 x 108/ml in HEPES-Tyrode buffer containing Ca2+ and Mg2+. Washed platelets were kept at room temperature (RT) for no longer than 4 h before use in flow assays. To induce platelet activation, washed platelets alone were incubated for 10 min at 37°C with thrombin (2 U/ml; Sigma) in the presence of the fibrin polymerization inhibitor GPRP-NH2 (2 mM; Sigma), henceforth referred to as thrombin-GPRP treatment. Previous work showed that inclusion of GPRP also prevents formation of homotypic platelet aggregates (25). RBCs were isolated from sodium citrate-anticoagulated blood, washed twice in Dulbecco's phosphate-buffered saline (DPBS) containing Ca2+ and Mg2+, and then resuspended to 40% (vol/vol) in DPBS, corresponding to normal physiological hematocrit.
Flow adhesion assays. LS174T or THP-1 cell interactions with HUVECs were quantified under simulated physiological flow conditions with a parallel plate flow chamber and a phase-contrast videomicroscopy-digital image processing system (5, 6). Attachment assays were performed by perfusing LS174T or THP-1 cells at a fixed concentration of 2 x 106/ml, with or without platelets, at the appropriate flow rates to obtain wall shear stresses of 0.52.0 dyn/cm2, thereby mimicking the fluid mechanical environment of the microcirculation and postcapillary venules (21).
The total numbers of primary and secondary tethering LS174T or THP-1 cells in a single x10 field of view (0.55 mm2) during the 5-min perfusion period were quantified by manually reviewing the videotaped experiments (6, 26). Primary tethers were defined as LS174T or THP-1 cells with or without adherent platelets that attached directly from flow to the HUVEC surface in the absence of interactions with previously captured cells. Secondary tethers were defined as those LS174T or THP-1 cells that first made contact with an already endothelium-adherent tumor cell before subsequently attaching to the endothelium downstream of the previously bound cell.
For inhibition experiments, HUVECs were pretreated for 30 min at 37°C, LS174T or THP-1 cells (107/ml) were pretreated for 15 min at RT, and platelets (2 x 108/ml) were pretreated for 10 min at 37°C with saturating concentrations of MAbs (1020 µg/ml) or nonpeptide small-molecule antagonists (100150 nM) before cell perfusion. In certain experiments, saturating concentrations of antibodies and/or antagonists were kept present in the flow buffer.
When washed RBCs were added to the perfusion buffer at the fixed, physiological RBC-to-platelet ratio of 25:1 and for a platelet concentration of 50 x 106/mlcorresponding to a platelet-to-tumor cell ratio of 25:1the resultant hematocrit was 10%. Selected experiments were also performed at physiological platelet concentration and hematocrit of
40%. Inclusion of washed RBCs led to an increase in the viscosity (µ) of the perfusion medium from 0.7 cP without RBCs to 0.9 and 1.2 cP at hematocrit levels of
10% and 40%, respectively, as measured in a cone and plate rheometer (18, 25). The volumetric flow rate was adjusted accordingly to obtain the desired wall shear stress (
) according to the standard equation
= 6µQ/wh2, where Q is the volumetric flow rate, w is the flow channel width (0.5 cm), and h is the flow channel height (250 µm) (21). When washed RBCs were added to the perfusion buffer, LS174T or THP-1 cells were not visible through the flowing RBCs under phase-contrast microscopy, thus necessitating the use of fluorescence videomicroscopy. To this end, LS174T or THP-1 cells were labeled for 1 h at 37°C with 4 µM carboxyfluorescein diacetate, succinimidyl ester (CFDA SE; Molecular Probes, Eugene, OR), washed once to remove excess dye, and resuspended in serum-free medium containing 0.1% bovine serum albumin. CFDA SE treatment does not affect the extent of cell binding (tethering) to HUVECs under flow (data not shown).
Cone and plate rheometry-flow cytometry assay.
LS174T cells, stained with 0.2 µM SNARF red fluorescent dye (Molecular Probes) according to the manufacturer's instructions, were suspended in DPBS containing Ca2+ and Mg2+ and stored at 4°C for no longer than 4 h before use in the rheometry assay. Washed platelets, treated with thrombin-GPRP as described above, and SNARF-stained LS174T cell suspensions were allowed to equilibrate separately to 37°C for 2 min. Thereafter, LS174T cells (1 x 106 cells/ml) and washed platelets, with or without washed RBCs (final Hct = 40%), were placed onto the stationary platen of a cone and plate rheometer (RS150; Haake, Paramus, NJ) to achieve the desired ratio of platelets to tumor cells (3:1). Specimens were subjected to a shear rate of 2,500 s1 for 60 s at 37°C. On termination of shear, aliquots were obtained by pipette, instantly fixed with 1% formaldehyde, labeled with a FITC-conjugated platelet-specific MAb directed against IIb
3 (5 µg/ml c7E3-FITC), and subsequently analyzed by flow cytometry (25). Three thousand SNARF-stained cell events were acquired to determine 1) the percentage of LS174T cells in heteroaggregates and 2) the population distribution of bound platelets to the LS174T cell surface, as previously described (25).
Confocal microscopy.
LS174T cells were labeled with 2 µM red fluorescent lipophilic membrane stain PKH26 according to the manufacturer's (Sigma) protocol, and platelets were similarly stained with 2 µM green fluorescent PKH67 (Sigma) (28). HUVECs were incubated with 10 µg/ml anti-ICAM-1 MAb for 30 min at 37°C before use in flow chamber assays. Previous work showed that ICAM-1 is not involved in adhesion of LS174T cells to TNF--stimulated HUVECs (6). Labeling with dyes did not affect cell tethering or adhesion compared with nonlabeled cells (data not shown). After the 5-min cell perfusion, slides were fixed for 1 h at RT in 4% paraformaldehyde in 100 mM cacodylate buffer (pH 7.4). Slides were washed and then incubated for 2 h with 10 µg/ml Alexa 633 goat anti-mouse IgG conjugate (Molecular Probes). After a final wash step, slides were mounted in antifade medium 1,4-diazabicyclo[2,2,2]octane (DABCO; Sigma) and observed on a Zeiss Meta 510 scanning confocal microscope.
Statistics. Data are expressed as means ± SE. Statistical significance of differences between means was determined by one-way ANOVA. If means were shown to be significantly different, multiple comparisons by pairs were performed by the Tukey test. Probability values of P < 0.05 were considered statistically significant.
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RESULTS |
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The number of secondary tethers also depended on platelet concentration. Indeed, the percentage of secondary tethers relative to total tethers increased progressively from background levels (<5%) without platelets to 60% at a platelet-to-LS174T cell ratio of 25:1. Similar observations were also made for the L-selectin-negative monocytoid THP-1 cells when perfused simultaneously with thrombin-activated platelets over HUVECs. An increase in total interacting cells (from 191 ± 14 to 577 ± 28 cells/mm2; n = 56) and amount of secondary tethering (Fig. 1C) was detected as the platelet-to-THP-1 cell ratio was increased from 0 to 25:1. However, at 0.8 dyn/cm2,
70% of total THP-1 cells attaching to HUVECs were a result of secondary tethering at a platelet-to-THP-1 cell ratio of 3:1 compared with
10% of total tethered LS174T cells at the same ratio (Fig. 1C).
The total amount of tumor cell tethering to HUVECs was also strongly dependent on wall shear stress, with a peak in total LS174T cells interacting with endothelium achieved at 0.50.8 dyn/cm2 in the presence of activated platelets (platelet-to-LS174T cell ratio of 25:1, or 50 x 106 platelets/ml; Fig. 3A). No platelet enhancement in tumor cell tethering was observed at 2.0 dyn/cm2 (Fig. 3A), consistent with a minimal occurrence of secondary tethers (Fig. 3B). This may be due to diminished primary tethering (67 ± 14 cells/mm2) that is below the "threshold" needed to observe secondary accumulation (140 cells/mm2 at 2 min; Fig. 1B). Nevertheless, a peak in secondary tethering, parallel to that of total tethering, was observed at 0.50.8 dyn/cm2 when shear stress was increased from 0.5 to 2 dyn/cm2 while holding the platelet-to-tumor cell ratio constant at 25:1 (Fig. 3B). It is noteworthy that in the presence of nonstimulated platelets secondary tethers did not occur and that the total amount of tethering was not significantly affected at any platelet concentration or shear stress, relative to platelet-free perfusions (data not shown).
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When LS174T cells were perfused with physiological concentrations of platelets (2 x 108/ml) and RBCs (Hct = 40%) at a constant platelet-to-LS174T cell ratio and a wall shear stress level of 0.8 dyn/cm2, secondary tethering was still observed (36 ± 4%; n = 2). In accord with these observations, the presence of RBCs (Hct = 40%) did not significantly disrupt the formation of heterotypic aggregates between thrombin-activated platelets and LS174T cells in shear flow (Table 1), which are critical for the secondary tethering process.
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DISCUSSION |
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Activated, but not nonactivated, platelets augmented LS174T and THP-1 cell adhesion to endothelium through the secondary tethering mechanism and not by bridging either LS174T or THP-1 cells to the target substrate (4-h TNF--stimulated HUVECs) under the conditions of this report, in contrast to previous investigations. For example, earlier studies examining the role of nonexogenously stimulated platelets in tumor cell adhesion to ECM components under flow conditions did not indicate secondary tethering as a possible adhesion pathway. Rather, tumor cells were often found in large platelet or platelet-leukocyte thrombi (13, 14), in which platelets deposited on ECM proteins supported firm adhesion of the cancerous cells (13, 14). Without platelets, the tumor cells were unable to attach to the ECM proteins under consideration. Together, these earlier findings suggest that platelets are capable of enhancing tumor cell binding by forming an adhesive bridge to the immobilized ligand. Along these lines Diacovo et al. (11) showed that TRAP-stimulated platelets adherent on leukocytes can bridge leukocytes to HEVs of peripheral lymph nodes in vivo when L-selectin function is blocked. No data regarding secondary tethering were reported. Furthermore, Theilmeier et al. (35) demonstrated that at moderately high shear rates (
400 s1) at which THP-1 cells alone cannot bind to stimulated HUVECs, addition of TRAP-activated platelets that became attached to THP-1 cells promoted tethering on endothelium. Again, no role for secondary tethering was identified, but the shear rate in that study was substantially higher than the optimal shear condition (0.8 dyn/cm2
100 s1) and the highest shear able to support LS174T cell adhesion (2.0 dyn/cm2
250 s1) in our investigation of tumor cell attachment to endothelium. Furthermore, the presence of activated platelets did not affect LS174T cell primary and secondary tethering to HUVECs at 2.0 dyn/cm2 (Fig. 3), even though platelets were clearly adherent on the endothelium-bound LS174T cells as determined by phase-contrast microscopy (unpublished observation). Aside from confocal microscopy observations that showed almost no platelet adhesion on the surface of endothelial cells, it is evident from the use of specific antagonists that interfere with function of receptors (i.e., P-selectin,
IIb
3-integrin, ICAM-1) mediating platelet binding to vascular endothelium that platelets are not involved in bridging LS174T and THP-1 cells to HUVECs. For instance, P-selectin MAb treatment failed to influence "primary" tethering of either cell type. Moreover, blockade of
IIb
3-integrin adhesive function abrogated secondary, not primary, tethering in LS174T experiments, yet no inhibition was observed on THP-1 cell interactions. These observations support the concept that platelets are only involved in secondary tethering.
Despite the controversy surrounding the physiological importance of secondary leukocyte tethering, the incidence of homotypic leukocyte tethers (secondary tethering) (2, 36) as a method of recruitment to sites of inflammation cannot be excluded. The possibility that tumor cell adhesion to the blood vessel wall can be similarly enhanced, albeit through a platelet-mediated secondary tethering mechanism, also deserves consideration. Although two-part perfusions of LS174T cells and platelets followed by LS174T cells alone revealed that platelets adherent on endothelium-bound LS174T cells are capable of supporting secondary tethering of free-flowing tumor cells via P-selectin and/or IIb
3 ligands, platelets adherent on tumor cells in bulk flow binding endothelium-attached LS174T cells through the appropriate counterreceptors or through platelet-platelet interactions may occur as well. However, the platelet-platelet scenario seems unlikely. The
IIb
3-integrin is necessary in platelet homotypic aggregation (21), but when its function was blocked by XV454, platelet-mediated THP-1 cell secondary tethering was unaffected (Fig. 5). Similarly, no inhibitory effect was detected on blocking platelet GPIb (Fig. 5). Therefore, THP-1-adherent platelets must interact with THP-1 cells directly (through platelet P-selectin binding to THP-1 cell PSGL-1). By extension, it can be concluded that platelet receptors most likely bind directly to ligands on the LS174T cells, rather than through platelet-platelet interactions.
Earlier studies in our laboratory (25, 27) suggested that colon carcinoma cell-platelet adhesion follows a multistep sequential process involving platelet P-selectin and IIb
3- integrin. In recruitment to sites of platelet deposition, platelet P-selectin is required for the efficient capture of free-flowing LS174T cells as well as rolling on the platelet monolayer whereas
IIb
3-integrin is required to convert the transient tethering events into firm adhesion (27). However,
IIb
3-integrin can also mediate the direct capture of some tumor cells from flow at 0.8 dyn/cm2 in the absence of any P-selectin contribution, perhaps because of the abundance of
IIb
3 molecules on the platelet surface after activation (21, 27). As the most plentiful platelet receptor with 50,00080,000 copies per platelet (8), receptor density and avidity may overcome the slower integrin binding kinetics that could otherwise impair
IIb
3-mediated tethering from flow (27). Therefore, it is possible that P-selectin and
IIb
3 may have overlapping roles in mediating the secondary tethering of LS174T cells, especially because P-selectin blockade is not completely effective in abolishing secondary tethering. This pathway is distinct from that of THP-1 cell secondary tethering, which is wholly P-selectin dependent and
IIb
3 independent.
The potential (patho)physiological relevance of the secondary tethering mechanism may also revolve around the influence of erythrocytes on platelet-tumor cell heterotypic interactions. Our rheometric-flow cytometric data clearly indicate that the presence of RBCs does not significantly interfere with the formation of stable platelet-LS174T heteroaggregates in shear flow. This finding is in accord with the observations that platelet-mediated secondary tethering indeed occurs in the presence of RBCs at both low (10%) and physiological (40%) hematocrit levels. The reduced levels of secondary tethering observed in the presence of RBCs may be explained by the "physical" barrier that the RBCs impose, which may interfere with the transient collisions of LS174T-adherent platelets and other free-flowing or endothelium-adherent LS174T cells (30) necessary for secondary tethering. Alternatively, RBCs may collide with free-flowing LS174T cells, thereby increasing the hydrodynamic dispersal forces between a free-flowing and an already adherent cell during the secondary tethering process (29, 30).
Cumulatively, the present work demonstrates a novel role for activated platelets in supporting secondary tethering of colon carcinoma cells, resulting in platelet-induced enhancement of cell recruitment to stimulated endothelium compared with platelet-free perfusions. This platelet P-selectin/IIb
3 integrin-mediated phenomenon may be physiologically relevant because it can take place in the presence of erythrocytes. Moreover, several reports indicate that platelet activation could be triggered in vivo by tissue factor constitutively expressed on the surface of many tumor cells, including colon carcinomas, by inducing thrombin production in the coagulation cascade (20, 31). Overall, these findings promote our understanding of the role of platelets in hematogenous spread of cancer, which could potentially lead to new strategies to combat metastasis.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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