Animals

All mice were on C57BL/6J background. GPIIb^-/- mice (αIIb-integrin)-deficient mice were generated as described previously [20]. Age- and sex-matched GPIIb^+/+ (WT) littermates served as controls. Animals were housed in specific pathogen free conditions in individually ventilated type III cages from TECNIPLAST (Hohenpeißenberg, Germany). Mice received standard chow from Altromin (Lage, Germany) and sterile tap water ad libitum. Appropriate enrichment (plastic-houses from TECNIPLAST, sterile pulp paper and coarsely litter) was provided. Physical condition of mice was monitored twice daily. A protocol approved by the Government of Bavaria for early euthanasia of potentially ill or moribund mice was in place. All experimental procedures on animals met the requirements of the German legislation on protection of animals and were approved by the Government of Bavaria, Germany.

Antibodies

For evaluation of melanoma surface molecule expression by flow cytometry, PE-conjugated rat anti-mouse αv-monoclonal antibody (mAb) (clone RMV-7), anti-β3-mAb (clone HM beta3.1) and rat IgG_2a-isotype control were obtained from BD Pharmingen (Heidelberg, Germany). anti-αIIb-mAb (clone MWReg 30) was obtained from eBioscience. FITC-conjugated mouse anti-human αvβ3-mAb (clone LM609) was purchased from Chemicon, mouse anti-human αIIb-mAb (clone P2) from Immunotec (Beckam Coulter, Krefeld, Germany) and IgG isotype control (X 0927, X 0928) from Dako (Glostrup, Denmark). For evaluation of platelet surface molecule expression by flow cytometry, anti-β3-mAb (BD Biosciences, clone 2C9.G2), anti-αIIb-mAb (eBioscience, clone MWReg30), GPIb (Emfret Analytics, clone X488), GPVI (Emfret Analytics, clone JAQ1), GPIX (Emfret Analytics, clone Xia.B4), anti-P-Selectin antibody (BD Biosciences, clone RB40.34), Annexin V (BD Pharmingen) and respective isotype controls were used. For fluorescence microscopy, polyclonal FITC-conjugated anti-GFP and goat rhodamine-conjugated anti-PE secondary antibody from Rockland (Gilbertsville, PA, USA), anti-ki67-PE (clone B56) from BD Biosciences (Cat. No. 556027, Germany), rabbit polyclonal anti-fluorescein/Oregon Green IgG (Molecular Probes, Karlsbad, CA, USA), rat anti-mouse αIIb-mAb (clone MWReg 30, BD Pharmingen), and anti-rat Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes) were purchased. For flow chamber experiments, c7E3 Fab (ReoPro) was obtained from Lilly (Bad Homburg, Germany).

Evaluation of platelet surface molecule expression

Expression of platelet surface molecules was investigated in washed murine platelets. Whole blood was obtained from GPIIb^+/+ and GPIIb^-/- mice by puncture of the inferior vena cava and anticoagulated with sodium citrate. Whole blood was washed and centrifuged in the presence of 0.5 μg prostaglandin. Washed platelets were resuspended in Tyrode’s buffer. Resting washed platelets were incubated with antibodies indicated above and surface expression was measured by flow cytometry. Surface P-Selectin- and AnnexinV/PS-exposure was determined in resting and Thrombin (0.05 U/mL, 10 min) stimulated platelets.

Cell culture

B16-D5 mouse melanoma cells, a poorly immunogenic subclone of the spontaneously arising B16-BL6 melanoma, were kindly provided by Dr. Winter (Franz Cancer Research Center, Earle A. Chiles Research Institute, Portland, OR) [25, 26]. αvβ3-positive M21 and αvβ3-negative M21L human melanoma cells were kindly provided by Prof. Dr. Haubner (Dep. of Nuclear Medicine, Medical University of Innsbruck, Austria) [27]. Cryo-preserved pooled primary human umbilical vein endothelial cells (HUVEC) were obtained from Promocell (Heidelberg, Germany) and cultured as described [28]. Cells were grown until confluence, and either left untreated (resting) or stimulated with 50 ng/mL TNF-α and 20 ng/mL IFN-γ for 20 hours before flow chamber experiments were performed.

Intravital microscopy

Mice were anesthetized by intraperitoneal application of Midazolam (10mg/kg body weight, Ratiopharm, Ulm, Germany), Medetomidin (1mg/kg, Pfizer, Karlsruhe, Germany) and Fentanyl (0,2mg/kg, Curamed Pharma, Munich, Germany). A polyethylene catheter was introduced into the left jugular vein. Upon tracheotomy mice were intubated and mechanically ventilated (MiniVent Typ 845, Hugo Sachs Electronics, March, Germany) at 120 strokes/minute and 150μl stroke volume of 0,5% isofluran-oxygen ratio [29]. Subsequently, a partial right thoracotomy with costal resection was performed to exhibit an oval lung field of approximately 1,5 x 2 cm². To minimize movements of the diaphragma, which readily conduct to the lung, the right phrenical nerve was anesthetized by local application of 1% Lidocaine (B. Braun, Melsungen, Germany). B16-D5 cells were labeled with 5-carboxyfluorescein diacetate succinimidyl ester (DCF) and adjusted to 1x10⁵ cells in 250μl Tyrodes buffer. DCF-tagged B16-D5 were intravenously infused in syngeneic mice on C57BL/6J background. For experiments conducted in vivo, GPIIb^-/- mice and their sex-matched GPIIb^+/+ littermates were handled as treatment pairs, thus receiving cells from the same preparation. In another set of experiments, 1.5x10⁸ isolated WT or GPIIb^-/- platelets were administered into GPIIb^-/- mice prior to B16-D5 application where indicated. Immediately and 1 hour after B16-D5 application the B16-D5 arrest in the pulmonary vasculature was monitored in vivo. Using confocal laser scanning fibre bundle microscopy [30] (Cellvizio^TM, Mauna Kea Technologies, Paris, France) we directly visualized the DCF-tagged tumor cells in vivo (S3 Fig). A flexible fibre bundle microprobe (MiniO Proflex microprobe, diameter 300μm, Mauna Kea) with a maximal optical penetration depth of 150μm was placed on the lung surface. The vasculature of the dorsal, ventral and basal pulmonary margin of each mouse was visualized in identical chronological order and direction. All images were taken during a ventilation pause following exhalation. Videos were recorded in real-time at a speed of 12 frames per second for subsequent analysis. To quantify melanoma retention in the lung, the number of retained tumor cell clusters was counted in the total range of the dorsal, ventral and basal right pulmonary margin from the recorded videos. To evaluate the size of accumulated tumor cell aggregates in the pulmonary vasculature, the first 10 events imaged at the dorsal margin were selected and their area was subsequently assessed using computer-assisted image analysis software (CapImage, Dr. Zeintl, Heidelberg, Germany).

Evaluation of B16-D5 metastasis formation

To determine the effect of GPIIb-IIIa mediated platelet action on metastasis formation in the lung, 1x10⁵ B16-D5 were infused into the jugular vein of GPIIb^+/+ (WT) or GPIIb^-/- mice. As described above, GPIIb^-/- mice and their sex-matched GPIIb^+/+ littermates were studied as treatment pairs. 10 days later, animals were euthanized by cervical dislocation and the number of metastatic nodules on the surface of the right lung was counted by blinded staff, and thereafter processed for histological analysis as described below. In addition, mRNA was isolated from the left lung of the same animal and pre-melanosomal protein (Pmel; Mus musculus 16756-Si: Silver) expression was quantified using Real-Time PCR (Quantitec Primer Assay, QT00102487, Qiagen, Hildesheim, Germany). Total mRNA was isolated with the RNeasy Midi Kit (Qiagen) following manufacturer’s instructions, and further reverse transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Darmstadt, Germany). Quantitative Real-Time PCR was performed with SYBR Green (Fermentas, Leon-Rot, Germany) as a fluorescent dsDNA-binding dye and Pmel expression was quantified by the ΔCt-Method after normalization to a housekeeping gene (beta-actin). All reactions were run as triplicates.

Histology

1) Histological analysis of GFP⁺ B16-D5 interaction with platelets: Lung specimens were obtained after injection of 0.5x10⁶ GFP⁺ B16 and incubated overnight at 4°C in a fixative containing 0.01M sodium periodate, 0.075M L-lysine and 1% paraformaldehyde. Thereafter, tissues were washed with 0.1M phosphate buffer, passed through sucrose gradient solutions and embedded in OCT compound (TissueTek®, Sakura, Netherlands). Cryostat sections (7 μm) were stained with the following reagents: 4',6-Diamidino-2-phenylindol (DAPI), rat anti-mouse GPIIb (CD41) and an Alexa Fluor 594-conjugated anti-rat antibody. To enhance the GFP-signal of B16-D5 we used a FITC-conjugated anti-GFP antibody (Rockland, Rockland, PA). Stained sections were mounted in Prolong Gold anti-fade mounting medium (Thermo Fisher Scientific, Waltham, MA).

2) Ki67 staining of lung specimen. After determining colonization, lungs were fixed in 10% neutral buffered formalin solution, dehydrated and embedded in paraffin. 20 sections of 3 μm thickness with 50 μm distance were generated from each lung. The sections were rehydrated and heat induced antigen retrieval was achieved by cooking the slides in citrate buffer (pH 6). Fc-blockade (BD Bioscience) was performed for 30 minutes. For sequential double immunofluorescence analysis sections were first treated with anti-ki67 PE (BD Biosciences) and a goat rhodamine-conjugated anti-PE secondary ab (Rockland), and then with an anti-HMB-45 mAb (Agilent, Santa Clara, CA) and a goat anti-mouse Alexa-488 secondary ab (Invitrogen™, Thermo Fisher Scientific), and DAPI. For detection of melanoma cells within the pulmonary tissue we applied the mouse anti-human antibody HMB-45 raised against the melanoma-specific marker Pmel. Human and mouse Pmel17/gp100 proteins have 79.7% amino acid sequence homology [31]. The mAb HMB-45 thus recognizes a shared epitope between both species, and murine B16 melanoma cell lines expressing Pmel17 demonstrated consistent staining with HMB-45 [32, 33]. Sections were investigated using a Leica DMRB epifluorescence microscopy.

Role of GPIIb for tumor growth

Wild-type and GPIIb-deficient mice were anesthetized and their back was shaved. 10⁵ B16-D5 cells in 200ul PBS were injected subcutaneously into the skin of the right flank. From day 14 after subcutaneous implantation, tumor diameters were measured every other day using a caliper. The tumor was explanted on day 21 post-injection and its weight was measured. The majority of mice from both genotypes developed a visible tumor within few days. Three mice of each genotype did not develop significant tumors during the whole duration of the experiment.

Assessment of platelet-tumor interactions under flow conditions

1) Interaction of human platelets with M21 melanoma cells. Whole blood was drawn from healthy donors and platelets were isolated as described [28]. All blood donors were volunteers who gave their free and informed written consent to this research study, which conforms to the ethical standards of the Declaration of Helsinki. Approval was obtained from the Technical University of Munich institutional review board for these studies. Flow chamber experiments were basically performed as reported earlier [28]. In brief, HUVEC were cultivated to confluency on glass cover slips for usage in a flow chamber, and either left untreated or co-stimulated with 50 ng/mL TNF-α and 20 ng/mL IFN-γ for 20 hours. HUVEC were perfused with DCF-labeled M21 or M21L at a shear rate of 1000/s for 10 minutes in the presence or absence of platelet-rich plasma or washed human platelets. To block GPIIb-IIIa receptor specific action, melanoma cells or the platelet-melanoma cell suspensions were preincubated with the monoclonal antibody abciximab (c7E3 Fab, 4 μg/mL; Lilly Deutschland, Bad Homburg, Germany) for 15 minutes. Tumor-endothelium interaction and tumor aggregate formation was studied in a flow chamber system mounted on the stage of an Axiovert 100 (Zeiss, Jena, Germany) microscope. All images were videotaped and subsequently evaluated offline using a computer-assisted image analysis program (Cap Image 7.1, Dr. Zeintl, Heidelberg, Germany). The number of firmly adherent melanoma cells was assessed by counting the cells that did not move or detach from the endothelial monolayer within 20 seconds. Further, the number of tumor cells within platelet-melanoma-aggregates was quantified.

2) Interactions of murine platelets with B16 melanoma cells. Whole blood was drawn from mice by cardiac puncture and platelets were isolated as described [34]. 10⁶ B16 cells were perfused together with 10⁸ washed murine platelets for 20min at 37C. Calcium (1mM), magnesium (1mM) and fibrinogen (Sigma, 75 μg/ml) were supplemented [35]. Perfusion was performed at 1000/s in a closed flow chamber system allowing recirculation of cells at continuous unidirectional and pulsatile flow (Ibidi, Martinsried, Germany). Platelet tumor aggregate formation was determined by flow cytometric analysis of platelet (glycoprotein Ib, CD42) positive cells (as compared to PE-isotype control) within the B16-population. Data was quantified on a FACS Calibur(Becton Dickinson, Franklin Lakes, NJ) and evaluated using FlowJo v10, LLCsoftware (Ashland, OR)

Determination of platelet-tumor cell aggregate formation in vitro

M21 melanoma cells were incubated for 15 minutes in the absence and presence of platelet-rich plasma and subsequently analyzed by fluorescence and electron microscopy. For fluorescence microscopy, probes were stained with fluorescent-labeled phalloidin and fluorescent antibodies directed against CD41 and fibrinogen. For electron microscopy (EM), cells were fixed in a sodium-cacodylate buffer (pH 7.4) with glutaraldehyde (2.5%) and tannin (0.02%). For scanning EM (SEM) fixed cells were plated on coverslips, and for transmission EM (TEM) probes were embedded in epoxyresin. Electron microscopy was performed as previously described [36].

Statistics

Prism 5.0 (GraphPad, San Diego, CA) was used as statistical software. Data are presented as means ± SEM unless stated otherwise. For in vivo experiments with matched treatment pairs data were analyzed using Student’s paired two-sample t-test. Other data was analyzed using the Tukey-test or one-way analysis of variance (ANOVA) with a Tukey posthoc test where adequate. A value of P < 0.05 was regarded as significant.

Platelet-melanoma interaction under flow conditions

Adhesion of tumor cells in the target vasculature is the initial step of metastatic seeding. Activated platelets have recently been shown to promote tumor cell adhesion to endothelial cells in vitro under venous flow conditions [8, 10]. However, in the in vivo setting, tumor cells are exposed to higher shear forces and are likely to encounter mostly non-activated platelets. Therefore, we first addressed whether unstimulated platelets–similar to collagen-activated platelets [8]–are also able to promote tumor cell adhesion, when exposed to capillary flow in vitro. Yet, when we applied shear conditions resembling those prevailing in the pulmonary microcirculation [37], only few M21 human melanoma cells, and even less M21L melanoma cells, lacking the major adhesion receptor αvβ3 [27], adhered to resting endothelial cells under flow (Fig 1A, left image and Fig 1B). Addition of non pre-activated platelet-rich plasma did not increase the number of adherent melanoma cells (Fig 1A, middle image and Fig 1B). Only when we co-stimulated endothelial cells with 50ng/mL TNF-α and 20ng/mL IFN-γ for 20 hours adhesion of melanoma cells was significantly increased (Fig 1B). Yet again the presence of platelets did not significantly increase, but rather reduced the adhesion of tumor cells to the endothelial monolayer (Fig 1B). Together, this suggests that non pre-activated platelets are dispensable for adhesion of single melanoma cells to endothelial cells under capillary flow conditions.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Platelet-tumor-endothelium interaction under flow.

a) Photomicrographs show platelet and M21 aggregate formation under flow (1000/s). Co-perfusion of platelets and M21 cells induced formation of aggregates which did not firmly adhere and are therefore only partly focused (middle picture; arrows indicate platelet-M21 aggregates). Aggregation is virtually abolished in the absence of platelets (left picture) or the presence of c7E3 Fab (right picture). Bars 0.2mm. Images were taken using a Zeiss Axiovert 100 microscope, 20x objective. b) HUVEC (resting or TNF-α/IFN-γ-stimulated) were perfused with M21 or αv-deficient M21L melanoma cells, that lack the expression of both αvβ3 and αvβ5 integrins, in the absence and presence of platelet-rich plasma (10min, shear rate 1000/s). Results are shown as number of firmly adherent M21 and M21L (n = 5; *P<0.05 for M21 vs. M21+PRP on activated HUVEC). c) Frequency of M21 and M21L aggregates in the absence and presence of platelets and c7E3 antibody. Results are given as number of melanoma cell-aggregates in % of all perfused tumor cells (n = 5; *P<0.001 for M21 in the absence vs. presence of platelets). d) Size distribution of M21 aggregates in the absence and presence of platelets and c7E3. e-g) M21 melanoma cells were incubated for 15 minutes in the absence and presence of platelet-rich plasma. e) Scanning electron microscopy indicates single melanoma cells in the absence (left picture) and melanoma cell aggregates in the presence of platelets (right picture). Bars, 5μm. f) Transmission electron microscopy indicates direct platelet-melanoma interaction. Bar, 1μm. g) Photomicrographs show representative fluorescence microscopy images of M21-platelet aggregates stained with phalloidin (red) and anti-GPIIb (CD41) mAb (green). Bars, 20μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. h-i) Analysis of mouse GPIIb^-/- or WT platelet interaction with B16 melanoma under flow conditions (1000/s). h) Percentage of platelet-positive B16 cells (n = 4; *P<0.05). i) Representative FACS contour plots indicate CD42-staining on B16 cells in the absence (control) and presence of GPIIb^+/+ or GPIIb^-/- platelets.

https://doi.org/10.1371/journal.pone.0172788.g001

Formation of platelet-rich tumor cell aggregates in vitro

Previous in vitro data have identified that platelets bind to melanoma cells in a process involving platelet GPIIb-IIIa, fibrinogen and tumor cell-expressed αvβ3 integrin [8]. We hypothesized that rather than promoting tumor cell adhesion, GPIIb-IIIa mediates the formation of platelet–melanoma cell aggregates. In a first step, we analyzed in vitro whether platelet–melanoma cell aggregate formation occurs under flow. We perfused M21 and M21L in the presence or absence of platelets over a confluent endothelial monolayer applying capillary flow conditions. We found that in the absence of platelets, melanoma cells circulated as single cells, while aggregates of M21 cells (~6% of perfused melanoma cells), or αvβ3-deficient M21L cells were barely observed (<3% of perfused melanoma cells) (Fig 1A, left image, 1C and 1D). In contrast, in the presence of platelets, aggregate formation of M21 was significantly increased; approximately 20% of all melanoma cells were now incorporated into aggregates (Fig 1A, middle image and Fig 1C and 1D). When we examined the size of aggregates, we found that the few aggregates which formed in the absence of platelets mostly consisted of two cells, whereas larger aggregates were rarely detected (Fig 1D). In contrast, when platelets were added to/co-perfused with M21 cells, the aggregate size significantly increased; in fact, the number of aggregates containing 3–4 cells was approximately 5-fold higher in the presence of platelets, when compared to M21 cells in the absence of platelets. We even frequently found large aggregates comprising >5 cells in the presence of platelets (Fig 1D). Further analysis of the melanoma cell aggregates by immunofluorescence and electron microscopy revealed that platelets physically participated in melanoma cell aggregation. We observed numerous platelets/platelet aggregates interspersed between the assembled melanoma cells (Fig 1E–1G). The heterotypic platelet-melanoma cell aggregation under flow required GPIIb-IIIa. Correspondingly, aggregate formation was strongly reduced in the presence of the antibody c7E3 Fab targeting both GPIIb-IIIa and the vitronectin receptor αvβ3 integrin (Fig 1A, right image, Fig 1C and 1D).

We added another set of experiments to determine whether in murine cells GPIIb integrin also plays a role in platelet-tumor aggregate formation under capillary flow conditions. To do so, we isolated platelets from GPIIb^+/+ and GPIIb^-/- mice and co-perfused them with murine B16-D5 melanoma cells in the presence of fibrinogen in a closed flow chamber system [35]. Consecutive quantification by flow cytometry analysis revealed that also in the murine system platelet-B16 aggregate formation was enhanced in the presence of GPIIb (Fig 1H and 1I). To test whether GPIIb-deficiency alters platelet surface receptor expression, flow cytometry was performed with isolated GPIIb^+/+ and GPIIb^-/- platelets. We found no difference in surface expression of glycoprotein GPIb-IX-V complex or GPVI receptor in GPIIb^-/- platelets compared to GPIIb^+/+ controls. Further no difference in P-Selectin or Annexin V expression could be detected upon thrombin activation (S2A–S2C Fig). Taken together, these findings implicate that under in vitro capillary flow conditions non pre-activated platelets do not promote adhesion of single melanoma cells to endothelial cells, but rather trigger the formation of platelet-tumor cell aggregates which is in part dependent on GPIIb-IIIa integrin. Flow chamber assays, however, are not capable of resembling the complex in vivo capillary flow conditions, such as alternating shear rates, tube diameters or variable site densities of endothelial adhesion molecules which all may influence melanoma cell adhesion and aggregation behavior [38, 39]. Hence, we designed mouse experiments to define whether platelet-melanoma cell aggregate formation also occurs in vivo and whether this mechanism is of any relevance for pulmonary metastasis.

Circulating platelet-melanoma aggregates form in vivo and are retained in the pulmonary vasculature

To evaluate intravascular conglomeration of melanoma cells, we infused 0.5 x 10⁶ GFP-expressing B16-D5 into the jugular vein of anaesthetized, spontaneously breathing WT mice. One hour after B16 application, citrated blood was carefully drawn from the right ventricle and lungs were harvested. Using flow cytometry, we determined numerous CD41-positive platelets attached to GFP⁺ B16-D5 in mouse whole blood (figure not shown), suggesting that platelet-induced melanoma-aggregate formation occurs under flow both in vitro and in vivo. When we examined the lungs of these animals one hour after infusion of GFP⁺ B16-D5 melanoma cells, we found large aggregates consisting of B16-D5 cells and platelets within the lung capillary network (Fig 2A). Thus, platelet-dependent aggregate formation in the blood and lungs is an early event after tumor cell infusion into mice in vivo.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Platelet-tumor-aggregate formation in vivo.

a) GFP-transfected B16-D5 melanoma cells were injected intravenously into wildtype mice. After 1 hour, lung tissue was obtained for immunofluorescence analysis. Photomicrographs show mouse lung tissue stained with antibodies directed against platelet GPIIb (CD41, red) and B16-D5 (GFP, green); nuclei were stained with DAPI (blue). Bars, 40μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x objective. b-d) Arrest of DCF-tagged B16-D5 melanoma cells was visualized in the pulmonary vasculature by intravital confocal videofluorescence microscopy (IVM) in GPIIb^+/+ and GPIIb^-/- littermate mice immediately and after 1 hour. b) Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- mice compared to its WT littermate (n = 5–6 experiments per group; *P<0.01 acute; **P<0.001 after 1 hour). c) Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- mice compared to its WT littermate (n = 5–6; *P<0.05 after 1 hour). d) Photomicrographs show representative IVM images obtained in GPIIb^+/+ and GPIIb^-/-. In GPIIb^+/+ mice, arrest of large multicellular aggregates is frequently observed (left). Arrest of DCF-tagged B16-D5 is visualized in precapillary vessels (right). Bars, 20μm. d e-f) GPIIb^+/+ (WT) or GPIIb^-/- platelets were injected into GPIIb^-/- mice just prior to administration of DCF-labeled B16-D5. IVM was performed immediately and after 1 hour. e) Number of metastatic events was quantified. Results are given as percentage of firmly adherent B16-D5 in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 4; *P<0.05). f) Size of B16-aggregates was quantified. Results are given as percentage of B16-aggregate size in GPIIb^-/- littermates receiving GPIIb^-/- platelets (n = 3; P = n.s.).

https://doi.org/10.1371/journal.pone.0172788.g002

Determination of melanoma cell-aggregate embolization in the pulmonary microcirculation using IVM

Based on the above findings we hypothesized that rather than adhesion of individual tumor cells, the platelet-dependent formation of melanoma cell-aggregates and their subsequent retention within the pulmonary circulation triggers efficient metastatic melanoma cell seeding. In order to address this hypothesis, we analyzed in vivo whether blood-borne tumor cell-platelet aggregates can indeed be trapped within the pulmonary microcirculation. We therefore directly visualized the recruitment of melanoma cells in the pulmonary vasculature using real-time in vivo confocal microscopy (IVM) of the mouse pulmonary vasculature (S3 Fig). DCF-labeled B16-D5 cells were administered into the jugular vein of anaesthetized and mechanically ventilated WT and GPIIb^-/- mice. Immediately after cell infusion, numerous melanoma cells accumulated in the WT lung vasculature. IVM revealed that B16-D5 melanoma cells are rarely recruited to the WT pulmonary circulation as single cells, but mostly accumulate in the form of multicellular aggregates captured in larger vessel (S1 Videos). In several occasions B16-D5 were entrapped at vascular bifurcations. In contrast, significantly less melanoma cells accumulated in pulmonary vessels of GPIIb^-/- mice within the first minutes after infusion (Fig 2B). Moreover, when we quantified the size of B16-D5 conglomerates, we found that there was not only a decrease in the overall number of retained aggregates but also that B16-D5 aggregates were significantly smaller in GPIIb^-/- mice compared to WT animals (Fig 2B and 2C). The difference between WT and GPIIb^-/- mice was even more pronounced 1 hour after B16-D5 administration (Fig 2B and 2C). This suggests that a substantial proportion of those melanoma cells initially retained in GPIIb^-/- mice was able to pass the pulmonary microcirculation after prolonged transit times.

GPIIb-IIIa has been considered to be expressed exclusively on cells derived from the megakaryocytic/platelet lineage. However, recent evidence suggests that also some tumor cell lines surface express GPIIb-IIIa [40, 41]. Nevertheless, when we performed flow cytometry we found that neither B16-D5 nor M21 melanoma cells expressed GPIIb (S1 Fig). In order to exclude that GPIIb-IIIa from additional (non-platelet, non-melanoma cell) sources contributes to melanoma cell-aggregate formation and retention in vivo, we performed an additional set of experiments in which GPIIb^-/- mice were treated with GPIIb^-/- or WT platelets prior to administration of DCF-labeled B16-D5. After 1 hour, IVM of the pulmonary vasculature was performed as described above. When WT platelets were infused into GPIIb^-/- mice we now observed an increase in number of tumor cells that accumulated in the pulmonary vasculature compared to GPIIb^-/- mice receiving GPIIb^-/- platelets (Fig 2E and 2F). This indicates that melanoma cell recruitment in the pulmonary microcirculation is mediated by platelets and depends on platelet GPIIb-IIIa. At the same time this set of experiments makes it unlikely that in GPIIb^-/- mice a pulmonary phenotype restricts tumor cell accumulation.

Taken together, our in vivo imaging revealed that platelet-melanoma cell coaggregation rather than adhesion of single melanoma cells is essential for their initial capturing in the pulmonary circulation. This mechanical entrapment of large platelet-tumor cell aggregates could—at least in part—explain the pulmonary tropism of malignant melanoma.

Role of platelet GPIIb for melanoma metastasis after 10 days

We next aimed to further dissect the relevance of platelet GPIIb-mediated events in hematogenic melanoma cell metastasis beyond the acute effects on tumor cell arrest. To do so, we intravenously infused B16-D5 cells into WT and GPIIb^-/- mice. Ten days later the right lungs were harvested and the numbers of metastases counted in situ. Unexpectedly, we observed that despite the defect in initial melanoma cell retention in GPIIb^-/- mice, the mutants were not protected from metastasis formation at later stages. In fact, the number of metastases was significantly lower in lungs of GPIIb^+/+ littermates compared to GPIIb^-/-mutants (Fig 3A–3C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Effect of platelet GPIIb on melanoma metastasis formation.

a)-c) B16-D5 melanoma cells were injected intravenously into GPIIb^+/+ or GPIIb^-/- littermate mice. After 10 days, metastasis formation was analyzed. a) Photomicrographs show representative lung images. Bars, 0.5 cm. b) Quantification of metastatic nodules in the lung (n = 5; *P<0.05). Boxes indicate median, whiskers indicate min and max values. c) Quantification of Pmel mRNA expression. Individual Pmel data normalized to beta-actin is indicated as 2^(-ΔCt) (n = 5; *P<0.05). Boxes indicate median, whiskers indicate min and max values. d-e) Pulmonary melanoma proliferation was determined by analyzing Ki67 expression in HMB-45-positive melanoma cells 10 days after intravenous seeding (n = 5; *P<0.05). Photomicrographs show fluorescence microscopy images of mouse lung tissue stained with antibodies directed against melanoma HMB-45 (green) and Ki67 (red); nuclei were stained with DAPI (blue). Images show 3μm optical sections. Left image, overview of lung tissue section; right image, magnification. Bars, 100μm. Images were taken using a Leica DMRB epifluoresence microscope, 20x and 40x objective. f) Effect of platelets on M21 mitogenesis in vitro. Serum-starved M21 cells were grown in the absence or presence of increasing concentrations of washed human platelet and BrdU uptake was quantified (*P<0.05, **P<0.01). g-h) Effect of platelet GPIIb on subcutaneous melanoma growth. g) B16-D5 cells were injected subcutaneously in the right flank of wildtype and GPIIb^-/- littermate mice and tumor growth surveyed using a digital scaliper. h) 21 days after tumor cell seeding tissues were explanted. Tumor weight was measured indicating no difference in subcutaneous tumor growth (P = n.s.).

https://doi.org/10.1371/journal.pone.0172788.g003

In order to further evaluate the tumor load ten days after B16-D5 application, we analyzed the expression of melanoma cell specific antigen Pmel17/silver in the lungs. In line with macroscopic findings, mRNA expression of Pmel17/silver revealed an attenuated tumor load in GPIIb^+/+ mice as compared to their GPIIb^-/- littermates (Fig 3C).

Taken together, these findings have three major implications: (1) a GPIIb-IIIa mediated platelet mechanism is essential to allow initial tumor cell retention in the pulmonary vasculature; (2) despite favoring initial melanoma cell retention, GPIIb-IIIa-mediated platelet activity suppresses subsequent tumor growth at later stages after initial seeding; (3) consistent with recent findings in the brain microcirculation [6] our present observations in the lung indicate that initial seeding and micrometastasis formation are not inevitably linked to efficient growth to macrometastases.

Reduced melanoma proliferation in the presence of platelets and GPIIb

To address this apparent discrepancy and to further evaluate our observation that a platelet GPIIb-dependent mechanism inhibits melanoma growth after initial seeding, we checked for melanoma cell proliferation rates 10 days after melanoma cell injection using Ki67 staining on lung sections. For identification of melanoma cells within the pulmonary tissue we used the antibody HMB-45 raised against the melanoma-specific marker Pmel. We found that in GPIIb^-/- mice significantly more melanoma cells were Ki67 positive compared to their GPIIb^+/+ littermates (Fig 3D and 3E, S4 Fig). This indicates that the presence of GPIIb suppresses tumor cell proliferation in our model of melanoma metastasis formation.

To address whether platelets can directly influence melanoma growth, we next performed cell culture experiments of melanoma cells in the presence or absence of washed platelets. We studied incorporation of the thymidine analog bromodeoxyuridine (BrdU) as a marker for DNA synthesis and cell proliferation. We found that M21 melanoma proliferation was significantly reduced by addition of isolated human platelets. Notably, the effect was dose-dependent as incubation with increasing platelet numbers resulted in greater inhibition of tumor growth (Fig 3F). Thus, platelets, which accumulated in high numbers around melanoma cells in vitro (Fig 1A–1I) and in vivo (Fig 2A), increased initial tumor cell arrest and metastasis formation in vivo (Fig 2B–2D), but negatively regulated tumor cell proliferation (Fig 3D–3F).

GPIIb does not impact on extravascular tumor growth

The experiments described above focused on hematogenic metastasis formation. To further assess the role of GPIIb in tumor growth, B16-D5 cells were injected subcutaneously in the dorsal skin of WT and GPIIb-deficient mice. Tumor size in both groups increased over time and the growth curves largely overlapped (Fig 3G). After 21 days, explant tumor weight was not significantly different between GPIIb^+/+ (0.551 ± 0.14 g) and GPIIb^-/- (0.608 ± 0.21 g) (Fig 3H). These results demonstrate that the effect of GPIIb was restricted to formation for hematogenic metastases.