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Keywords:

  • lung cancer;
  • platelet microvesicles;
  • exosomes;
  • metastasis;
  • matrix metalloproteinases

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

The role of platelets in tumor progression and metastasis has been recognized but the mechanism of their action remains unclear. Five human lung cancer cell lines (A549, CRL 2066, CRL 2062, HTB 183, HTB 177) and a murine Lewis lung carcinoma (LCC) cell line (for an in vivo model of metastasis) were used to investigate how platelet-derived microvesicles (PMV), which are circular fragments shed from the surface membranes of activated platelets, and exosomes released from platelet α-granules, could contribute to metastatic spread. We found that PMV transferred the platelet-derived integrin CD41 to most of the lung cancer cell lines tested and stimulated the phosphorylation of mitogen-activated protein kinase p42/44 and serine/threonine kinase as well as the expression of membrane type 1-matrix metalloproteinase (MT1-MMP). PMV chemoattracted 4 of the 5 cell lines, with the highly metastatic A549 cells exhibiting the strongest response. In A549 cells, PMV were shown to stimulate proliferation, upregulate cyclin D2 expression and increase trans-Matrigel chemoinvasion. Furthermore, in these cells, PMV stimulated mRNA expression for angiogenic factors such as MMP-9, vascular endothelial growth factor, interleukin-8 and hepatocyte growth factor, as well as adhesion to fibrinogen and human umbilical vein endothelial cells. Intravenous injection of murine PMV-covered LLC cells into syngeneic mice resulted in significantly more metastatic foci in their lungs and LLC cells in bone marrow than in control animals injected with LCC cells not covered with PMV. Based on these findings, we suggest that PMV play an important role in tumor progression/metastasis and angiogenesis in lung cancer. © 2004 Wiley-Liss, Inc.

Upon activation, eukaryotic cells shed components of their plasma membranes into the extracellular space. These circular fragments, known as microvesicles (MV) or microparticles,1, 2, 3, 4 have been detected in various biologic fluids including peripheral blood (PB).5, 6, 7, 8, 9, 10 MV contain numerous proteins that are similar but not identical to proteins present in the membranes of the cells from which they originate. Analysis of the released MV revealed marked differences in their composition depending upon the process of their formation.7, 8 Recently, we and others have postulated that MV are important signaling molecules in intercellular crosstalk.11, 12, 13, 14 This communication between cells may perhaps have developed very early in the course of eukaryocytic evolution, before soluble mediators emerged.

It has been well documented that activated PB platelets release MV.8, 15 Platelet-derived microvesicles (PMV; also known as platelet-derived microparticles or PMP) are formed after stimulation of platelets with agonists such as thrombin and collagen or after their exposure to high-stress shear forces.7, 16, 17, 18, 19 PMV express several platelet-endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib, IaIIa and P-selectin (CD62P).11, 12 They may also contain bioactive lipids including sphingosine 1-phosphate (S1P) and arachidonic acid (AA).13, 20, 21, 22 The interaction of PMV with target cells was shown to trigger various biologic responses such as activation of endothelium, stimulation of cytokine and tissue factor expression by endothelial cells19, 23, 24 and chemotaxis of monocytic cells.3, 19 Recently, we reported that PMV regulate proliferation, survival and adhesion of human normal and malignant hematopoietic cells.11, 12 They may also play a role in HIV infection; for example, we have reported that PMV transfer the HIV-entry co-receptor CXCR4 to the surface of CXCR4-negative cells, and even infectious HIV particles inside PMV may enter cells in a “Trojan horse” type of mechanism.14, 25

PMV released by activated platelets are of 2 types, cell-surface membrane vesicles and exosomes, both of which can be isolated by differential gradient centrifugation.7 The PMV that are released from surface membranes are generated in a calcium flux-calpain-dependent manner and are relatively large (100 nm–1μ m).26 In contrast, exosomes tend to be much smaller (30–100 nm) and their release results from fusion of platelet α-granules with the plasma membrane, where they exist as intraluminal membrane-bound vesicles. After exocytosis, the exosomes enriched in the internal vesicles are released into the extracellular space.4 The main components of PMV are proteins and lipids, while carbohydrates in association with glycolipids or glycoproteins constitute about 10%.4, 7 The density of PMV appears to be directly related to the relative protein content, and many of the biochemically active components of the membrane consist of receptors, adhesion molecules, transporters and enzymes including matrix metalloproteinases (MMPs).

MMPs constitute a large family of proteolytic enzymes known to play a role in cancer progression, angiogenesis and metastasis.27, 28, 29, 30 The contribution of MMPs to the malignant process derives from their ability to degrade substrates such as extracellular matrix components as well as a variety of nonmatrix substrates (e.g., certain cytokines, chemokines and growth-factor receptors as well as adhesion molecules). Increased levels of MMPs have been demonstrated in various types of cancer, and a positive correlation has been shown between soluble and membrane-type (MT) MMPs and lung cancer progression and metastasis.30, 31, 32

In our study, we hypothesized that PMV could promote the growth and metastatic potential of lung cancer cells. We selected 5 human lung cancer cell lines as an in vitro experimental model and investigated the transfer of platelet-derived surface molecules by PMV to cancer cell membranes. We also examined the direct effect of PMV on activation of signal transduction pathways, chemotaxis, cell proliferation, expression of angiogenic factors (including MMPs), chemoinvasion and adhesion. Further, we employed murine Lewis lung carcinoma (LLC) cells to evaluate the in vivo effects of PMV on the metastatic behavior of cancer cells. Our results indicate conclusively that PMV promote the growth and metastasis of lung cancer cells by directly stimulating them and transferring platelet-derived molecules to their surfaces.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Human and murine PMV and exosomes

Human PB platelets were isolated from healthy volunteer donors who had given informed consent; the protocols used were approved by the institutional review board of the University of Louisville. Murine platelets were obtained from C57BL/6 mice, as previously described.11 The platelets were activated by thrombin (0.1 U/mL) and collagen (4 μg/mL) for 30 min at 37°C with stirring and centrifuged twice at 2,000g for 15 min at 4°C. The PMV-enriched supernatants were collected, centrifuged at 24,000g for 1 hr at 4°C and the pellets washed and resuspended in HEPES buffer, pH 7.4. The PMV were characterized by staining with phycoerythrin (PE)-conjugated anti-human antibodies against αIIbβ3 (Coulter-Immunotech, Marseille, France), P-selectin (CD62) (Becton Dickinson, San Jose, CA) and CXCR4 (Becton Dickinson Pharmingen, San Diego, CA). PMV were fixed in 1% paraformaldehyde prior to flow-activated cell sorting (FACS) analysis using the FACScan (Becton Dickinson, San Jose, CA). Smaller microvesicles enriched in exosomes were separated from larger ones by more vigorous ultracentrifugation (100,000g for 60 min at 4°C). Microvesicles enriched in exosomes were washed once and resuspended in HEPES buffer, pH 7.4. The concentrations of both the PMV and the exosomes were estimated by Bradford assay. As it has been reported that the concentration of PMV in peripheral blood as measured by protein concentration is about 5 μg/mL5 and can be elevated up to 10 times in cancer patients,53 the doses of PMV we employed in the experiments described below were within the clinically relevant range.

Cell lines

Human lung cancer cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD). CRL 2062 and CRL 2066 are derived from small-cell lung cancers, HTB 177 and HTB 183 from large-cell lung cancers and CCL 185 (A549) from a highly metastatic human lung carcinoma. A murine cell line CRL 1642 (Lewis lung carcinoma, LLC) derived from C57BL mice was also purchased from ATCC. All cell lines were maintained in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT).

FACS analysis

To demonstrate the presence of PMV on the surface of target cells, lung cancer cells incubated with PMV were stained with PE-anti-CD41 antibody (Coulter-Immunotech) and analyzed by FACS. As isotype controls, we employed PE-goat-anti-mouse antibodies.

Phosphorylation of intracellular pathway proteins

Western blot analysis was performed on protein extracts from cells as described33, 34, 35, 36, 37 after the lung cancer cells were stimulated with PMV or exosomes (30 μg/mL) for 5 or 10 min at 37°C. Phosphorylation of serine/threonine kinase AKT and p44/42 mitogen-activated protein kinase (MAPK) was detected by protein immunoblotting using mouse monoclonal 44/42 phospho-specific MAPK antibody and rabbit phospho-specific polyclonal antibodies (all from New England Biolabs, Beverly, MA) for each of the remainder, with horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (Ig)G or goat anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) as secondary antibody, as described.33, 34, 35, 36, 37 Equal loading in the lanes was evaluated by stripping the blots and reprobing them with the appropriate monoclonal or polyclonal antibodies: p42/44 anti-MAPK antibody clone 9102 and anti-AKT antibody clone 9272 (Santa Cruz Biotechnology). The membranes were developed with an ECL reagent (Amersham Life Sciences, Little Chalfont, UK), dried and exposed to film (HyperFilm, Amersham).

Chemotaxis

Lung cancer cells were resuspended in RPMI with 0.5% bovine serum albumin (BSA, Sigma, St. Louis, MO) and equilibrated for 10 min at 37°C. Prewarmed serum-free medium containing PMV or exosomes (30 μg/mL) was added to the lower chambers of a Costar Transwell 24-well plate (Costar Corning, Cambridge, MA). Transwell membrane inserts covered with 50 μL of laminin (20 μg/mL) were placed between the upper and lower chambers. Cells were seeded into the upper chambers at a density of 1 × 105 in 100 μL. After 24 hr the cells that had transmigrated were counted either on the lower side of the membrane or on the bottom of the transwell. The results are presented as a migration index (the ratio of the number of cells migrating toward the medium containing PMV to the number of cells migrating toward the medium alone).

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)

For analysis of mRNA levels of cyclins A2, D1, D2, D3, E, vascular endothelial growth factor (VEGF), interleukin (IL)-8 and hepatocyte growth factor (HGF), total mRNA was isolated from cells with the RNeasy Mini Kit (Qiagen, Valencia, CA). The mRNA was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA) as described.38 Detection of cyclins A2, D1, D2, D3, E, VEGF, IL-8, HGF and β-actin mRNA levels was performed by real-time RT-PCR using an ABI PRISM® 7000 Sequence Detection System (ABI, Foster City, CA). A 25 μL reaction mixture contained 12.5 μL SYBR Green PCR Master Mix and 10 ng of cDNA template (Table I). Primers were designed with Primer Express software. The threshold cycle (Ct), i.e., the cycle number at which the amount of amplified gene of interest reached a fixed threshold, was determined subsequently. Relative quantitation of mRNA for cyclins A2, D1, D2, D3, E, VEGF, IL-8 and HGF mRNA expression was calculated with the comparative Ct method. The relative quantitation value of target, normalized to an endogenous control β-actin gene and relative to a calibrator, is expressed as 2-ΔΔCt (fold difference), where ΔCt = Ct of target genes (cyclins A2, D1, D2, D3, E and VEGF, IL-8, HGF) − Ct of the endogenous control gene (β-actin), and ΔΔCt = ΔCt of samples for target gene − ΔCt of the calibrator for the target gene.

Table I. Real-Time PCR Primers
 Primers (5′–3′)
SenseAntisense
Cyclin A2CCT GCA TTT GGC TGT GAA CTA CTGC CCA CAA GCT GAA GTT TTC
Cyclin D1GCA TGT TCG TGG CCT CTA AGA TCGG TGT AGA TGC ACA GCT TCT C
Cyclin D2TTC TTC TTC CAA ATG CAG TTC ATTAGT TTT GGT TAT GGC CCA TAG C
Cyclin D3CAG CGC CTT TCC CAA CTC TAAAA AGG AAT GCT GGT GTA TGT ATC C
Cyclin ETGC AGA GCT GTT GGA TCT CTG TGCC GAA GCA GCA AGT ATA CCA T
VEGFCAC CCA TGG CAG AAG GAG GAGGT CTC GAT TGG ATG GCA GTA G
IL-8CTG GCC GTG GCT CTC TTGTTA GCA CTC CTT GGC AAA ACT G
HGFCTC ACA CCC GCT GGG AGT ACTCC TTG ACC TTG GAT GCA TTC
β-actinGGA TGC AGA AGG AGA TCA CTGCGA TCC ACA CGG AGT ACT TG

To avoid the possibility of amplifying the contamination of DNA, (i) all the primers for real-time RT-PCR were designed with an intron sequence inside the cDNA to be amplified; (ii) reactions were performed with appropriate negative controls (template-free controls); (iii) a uniform amplification of the products was rechecked by analyzing the melting curves of the amplified products (dissociation graphs); (iv) the melting temperature (Tm) was 57–60°C and the probe Tm was at least 10°C higher than the primer Tm; and (v) gel electrophoresis was performed to confirm the correct size of the amplification and the absence of unspecific bands.

Gel-based RT-PCR

To evaluate gene expression for MMPs and tissue inhibitor of metalloproteinase (TIMP)-2 in lung cancer cell lines, total RNA was extracted as described previously.39, 40 The conversion of mRNA to cDNA was carried out using avian myeloblastosis virus-reverse transcriptase (AMV-RT), and PCRs were carried out following the “primer dropping” method. Sequences for human MMP-2, MMP-9, MT1-MMP and TIMP-2 were obtained from Genbank (Los Alamos, NM) and were used to design primer pairs as described by us previously.39

Zymography and Western blot

MMP-2 and MMP-9 activities were evaluated by zymography as previously described,40, 41 and MT1-MMP level was examined using Western blot. Briefly, A549 lung cancer cells (2 × 106/mL) were pre-incubated in serum-free media in the absence (control) or presence of PMV (30 μg/mL) for 48 hr at 37°C, 5 % CO2, and the cell-conditioned media were collected and analyzed. For MT1-MMP immunoblotting, the cell-conditioned media were separated under denaturing conditions in a 10% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. After blockage overnight at 4°C with 5% fat-free dried milk in Tris-buffered saline and 0.05% Tween 20, the membrane was probed with a specific monoclonal antibody directed against the catalytic region of human MT1-MMP (clone 114-6G6, Chemicon International, Temecula, CA) for 2 hr at room temperature. The membrane was further probed with a secondary antibody (goat anti-rabbit, horseradish peroxidase-conjugated IgG) to visualize the bands. Chemiluminescence detection was performed using the ECL system (Amersham Life Sciences) and the Fluor-S MAX2 Multiimager and the Quantity One version 4.3.1 software (Bio-Rad).

Chemoinvasion assay

As A549 and HTB-177 cell lines exhibited upregulation in MMP-9 and MT1-MMP expression in the presence of PMV, respectively, we evaluated their invasive potential in a trans-Matrigel chemoinvasion assay as described.33, 40, 41 The lower chambers contained media supplemented with PMV (30 μg/mL) or media only (control). The cells pre-incubated with PMV, or not, were loaded onto the upper compartments (3 × 105 cells/chamber) and incubated (at 37°C, 5% CO2) for 48 hr. Cells that invaded the Matrigel were evaluated on the undersides of filters after fixation with 11% glutaraldehyde (Sigma, Oakville, Canada) and staining with 1% crystal violet. Three random fields were selected for microscopic count at 20× magnification (Leitz Diavert, Ottawa, Canada). To confirm the role of MMPs in the Matrigel invasion by lung cancer cells, the cells were pre-incubated with 0.5 mM of the MMP inhibitor o-phenanthroline (Sigma) for 30 min prior to loading onto the upper compartments of chambers, and the experiment was carried out as before. The viability of the cells was assessed by trypan blue staining and found not to be significantly decreased compared to cells not incubated with o-phenanthroline. The chemoinvasion assay was performed 3 times using at least 5 chambers for each condition.

Cell proliferation assay

A549 cells were cultured in serum-free medium supplemented with 0.5% BSA in the presence of PMV or exosomes (30 μg/mL) for 72 hr. Cells were cultured in 96-well plates at a concentration of 0.2 × 105 per well at 37°C in a fully humidified atmosphere supplemented with 5% CO2. The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was used according to the manufacturer's protocol to determine the number of viable cells.

Adhesion to fibrinogen and to human umbilical vein endothelial cells (HUVEC)

Cells were labeled with calcein-AM (Molecular Probes, Eugene, OR) for 30 min in medium containing 0.5% BSA, washed twice and incubated for 30 min with PMV or exosomes (30 μg/mL). Subsequently, cell suspensions (1 × 105 cells/100 μL) were applied to 96-well microtiter plates covered with fibrinogen (10 μg/mL) or HUVEC activated by tumor necrosis factor (TNF-α) and incubated for 3 hr at 37°C. After incubation, the plates were washed 4 times to remove nonadherent cells, and the number of adherent cells was estimated by measuring fluorescence in a spectrofluorimeter as described.33, 42

In vivo model of metastasis

Five- to 6-week-old female C57BL/6 mice (National Cancer Institute) were injected intravenously with 104–105 LLC cells that had been incubated or not with murine PMV. Incubation was done in 100 μL of phosphate buffer saline (PBS) with 0.5% BSA, 1 mM CaCl2 for 30 min at 37°C and at a PMV concentration of 300 ng/mL. After 3–4 weeks, the mice were killed and their organs (lungs, liver, brain) and femur bones were collected. The organs were fixed in ethanol and the bones in EFA buffer (ethanol, formamide, acetic acid) followed by decalcification in nitric acid solution. The number of metastatic foci was scored in the organs using a magnifying glass, and the number of tumor cells in the bone marrow (BM) was evaluated after hematoxylin-eosin staining of BM slides by counting the lung cancer cells visible under 670× magnification in several randomly chosen BM areas.

Statistical analysis

Arithmetic means and standard deviations of our data were calculated on a Macintosh computer PowerBase 180, using Instat 1.14 (GraphPad, San Diego, CA) software. Statistical significance was defined as p < 0.05. Data were analyzed using the Student t-test for unpaired samples.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

PMV transfer platelet-derived CD41 to the surface of lung cancer cells

We had demonstrated previously that PMV transfer platelet-derived surface receptors to various normal and malignant hematopoietic cells.11, 12, 14 Hence, we wished to determine whether the same was true for lung cancer cells. All 5 human lung cancer cell lines studied became CD41-positive when pre-incubated with PMV (Fig. 1).

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Figure 1. Binding of PMV to human lung cancer cells. Lung cancer cell lines were incubated with PMV and expression of CD41 was evaluated by FACS analysis; shaded curves represent isotype controls. Data are from a representative experiment, which was done 3 times with similar results.

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PMV induce phosphorylation of MAPK p42/44 and AKT in lung cancer cells

Since all lung cancer cell lines interacted with PMV, we next investigated whether this interaction activated signaling pathways of proliferative responses (MAPK p42/44 and AKT Ser473) in these cells as we had observed for human hematopoietic cells.12 We found that stimulation of lung cancer cells with PMV induces the phosphorylation of MAPK p42/44 and AKT in all cell lines tested (except in HTB 183 for AKT phosphorylation) (Fig. 2).

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Figure 2. Phosphorylation of MAPK p42/44 and AKT by PMV in A549, HTB 177, HTB 183, CRL 2062 and CRL 2066 human lung cancer cell lines. In each panel: lane 1, quiescent lung cancer cells; lane 2, cells stimulated for 5 min; lane 3, cells stimulated for 10 min by PMV (30 μg/mL). The experiment was done twice with similar results. A representative study is shown.

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PMV chemoattracted 4 of 5 lung cancer cell lines tested

Since we had previously observed that PMV chemoattract various malignant hematopoietic cell lines,12 we next investigated whether PMV can induce chemotaxis of human lung cancer cells as well. PMV at a dose of 50 μg/mL chemoattracted A549 cells 7-fold, HTB 177 cells 5-fold, HTB 183 cells 2-fold and CRL 2062 cells 2.5-fold more than the control (migration toward media only). CRL 2066 was the only cell line not chemoattracted to PMV (Fig. 3).

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Figure 3. Chemotaxis of lung cancer cells to PMV. A549, HTB 177, HTB 183, CRL 2062 and CRL 2066 lung cancer cells were resuspended in 0.5% BSA + RPMI and placed in the upper chambers of transwells. Transwell membranes were covered with laminin. The lower chambers contained medium with 10 (low-dose) or 50 (high-dose) μg/mL of PMV. After 24 hr the cells on the lower portion of the transwell membrane were stained and counted under an inverted microscope. The experiment was done 3 times with similar results. A representative study is shown. *p < 0.0001 indicates statistical significance relative to the control.

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PMV stimulate MMP expression and chemoinvasion

Extensive experimental data show that MMPs contribute to cancer progression and metastasis.27, 28, 29, 30 We evaluated whether the 5 human lung cancer cell lines studied express MMPs (MMP-2, MMP-9 and MT1-MMP) and whether PMV stimulate their expression. As tissue inhibitor of metalloproteinase (TIMP)-2 is crucial for proMMP-2 activation, we also examined the expression of this transcript. We found that the highly metastatic human lung cancer cell line A549 is the only 1 of the 5 studied in which all 4 transcripts, MMP-9, MMP-2, MT1-MMP (Fig. 4a,d) and TIMP-2 (data not shown), were found. Zymograms confirmed the presence of MMP-9 and MMP-2 in media conditioned by this cell line (Fig. 4b). PMV stimulated MMP-9 mRNA expression in A549 cells (2.9-fold) and proMMP-2 activation. PMV also significantly stimulated MT1-MMP mRNA expression in HTB 177 cells (5.7-fold), which translated into an immense increase in protein level as shown by Western blot (Fig. 4e). Slight upregulation of MT1-MMP mRNA expression was found in A549 (1.2-fold) and CRL 2062 (1.5-fold) cells, and all cancer cell lines expressed mRNA for TIMP-2 (not shown).

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Figure 4. Effect of PMV on MMP expression and trans-Matrigel chemoinvasion. (a–c) Expression of mRNA for MMP-9 and MMP-2 in 5 human lung cancer cell lines studied without (c, control) and after stimulation with 30 μg/mL PMV (+ P) (a); secretion of MMP-2 and MMP-9 by A549 cells without (control) and after PMV (+ PMV) stimulation (30 μl/mL) was evaluated by zymography (b); and chemoinvasion of A549 cells is increased after pre-incubation of cells with 30 μg/mL PMV (p = 0.02) and is inhibited by o-phenanthroline (0.5 mM) as evaluated in Matrigel assay (c). (d–f) Expression of mRNA for MT1-MMP in 5 human lung cancer cell lines studied without (c, control) and after stimulation with 30 μg/mL PMV (+ P) (d); protein expression of MT1-MMP by HTB 177 cells without (control) and after PMV (+ PMV) stimulation was evaluated by Western blot (e); and chemoinvasion of HTB 177 cells is increased after pre-incubation of cells with 30 μg/mL PMV (p < 0.001) and is inhibited by o-phenanthroline (0.5 mM) as evaluated in Matrigel assay (f). Media with 30 μg/mL PMV only (no-cells control) were also evaluated in the zymogram and Western blot.

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To evaluate the effect of PMV on the invasive potential of tumor cells, we selected A549 and HTB 177 cell lines, which showed the strongest upregulation in MMP expression in the presence of PMV and employed an in vitro assay of chemoinvasion across a reconstituted basement membrane barrier (Matrigel). We eliminated any concentration gradient by putting equal concentrations of PMV in the top and bottom chambers as we had shown that A549 and HTB 177 cells are most strongly chemoattracted to PMV (as shown in Fig. 3). We found significantly stronger chemoinvasion for both cell lines after incubation with PMV, which was abrogated by o-phenanthroline, a widely used metalloproteinase inhibitor, indicating that MMPs are involved in this process (Fig. 4c,f).

PMV enhance proliferation of A549 cells and upregulate cyclin D2 expression

Because we found that MAPK p42/44 and AKT were both strongly phosphorylated in A549 cells (Fig. 2), we selected this cell line for further studies. Since phosphorylation of MAPK p42/44 and AKT is essential for cell proliferation, we sought to determine whether PMV influence the proliferation of A549 cells. We found that both PMV and exosomes strongly stimulated the proliferation/survival of A549 cells in serum-free cultures, as shown by the MTT assay (Fig. 5).

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Figure 5. PMV increase proliferation of A549 cells. A549 cells were cultured in serum-free medium for 72 hr in the absence or presence of MV or exosomes (30 μg/mL) (n = 3). The effect of PMV and exosomes is compared to the growth of these cells in medium supplemented with 10% FBS. The MTS cell proliferation assay was done 3 times in quadruplicate with similar results. * p < 0.001 as compared to the control (0.5% BSA).

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Next, to elucidate the molecular mechanisms responsible for this effect, we evaluated changes of expression of mRNA for cyclins A2, D1, D2, D3 and E in A549 cells cultured for 24 hr in serum-free medium and then stimulated for 3–48 hr by PMV or exosomes. We found that while mRNA for cyclin A2, D1, D3 and E remained unchanged, the expression of mRNA for cyclin D2 was upregulated more than 6-fold and 10-fold after stimulation for 3 hr by exosomes and PMV, respectively (data not shown). At the same time, however, we did not observe any changes in expression of mRNA for apoptotic effectors bcl-2, Bax and bad (data not shown).

PMV modulate the adhesiveness of A549 cells to fibrinogen and HUVEC

Since we showed that PMV transfer CD41, which is important for platelet adhesion to fibrinogen or endothelium, to the surface of lung cancer cells (Fig. 1), we investigated whether A549 cells covered by PMV also adhere better to these substrates. Figure 6 shows significantly increased adhesion of A549 cells pre-incubated with PMV to both fibrinogen (Fig.6a) and HUVEC (Fig. 6b) compared to cells not pre-incubated (control).

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Figure 6. Adhesion of A549 cells covered with PMV to fibrinogen (a) and HUVEC (b). Calcein-AM-labeled A549 cells were not covered (control) or covered (PMV) with PMV and subsequently layered over fibrinogen (a) or HUVEC (b). The number of adherent cells was measured by spectrofluorimetry (relative fluorescence intensity) and microscopic analysis. Data from 3 separate experiments are pooled together and means ± SD are shown.

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PMV increase the metastatic potential of murine LLC cells

Since PMV bind to human lung cancer cells (Fig. 1), stimulate expression of MMPs (Fig. 4) and increase adhesion to endothelium and fibrinogen (Fig. 6), we asked whether lung cancer cells covered by PMV exhibit enhanced metastatic potential in vivo. To answer this question, we evaluated the influence of PMV on the metastatic behavior of murine LLC cells in vitro and in vivo. Our data show that PMV regulate several processes related to metastasis (Fig. 7). PMV transfer CD41 integrin to murine LLC cells (Fig. 7a), chemoattract them strongly (about 8-fold increase relative to control, Fig. 7b), stimulate their proliferation (Fig. 7c) and induce phosphorylation of MAPK p42/44 and AKT (Fig. 7d). Subsequently, in in vivo experiments, LLC cells covered and not covered with PMV were injected intravenously into syngeneic mice, and 2–3 weeks later metastatic foci were evaluated in these animals. We found that mice transplanted with LLC cells covered with PMV had significantly more macroscopically visible metastatic foci in their lungs than control animals transplanted with cells not covered with PMV. Accordingly, in 2 independent experiments, the weights of lungs from mice transplanted with PMV-covered LLC cells were 42 ± 7% greater than those of control animals (n = 12). This corresponded to an increase in the number of macroscopic metastases in the lungs of mice transplanted with PMV-covered LLC cells compared to controls (11 ± 3 vs. 6 ± 2, respectively). Moreover, we found that animals receiving PMV-covered LLC cells had more of these cells in their bone marrow cavities (Fig. 7e,f). On average, 18 ± 4 LLC cells were observed in the bone marrow sections derived from mice transplanted with PMV-covered LLC cells compared to 7 ± 2 in control mice. This observation agrees with our finding of increased adhesion to fibrinogen and endothelium of PMV-covered lung cancer cells and corroborates the evidence of the role of PMV in the metastatic process.

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Figure 7. Effect of PMV on murine LLC cells. (a) Binding of PMV to LLC. LLC cells were incubated with PMV and CD41 expression was evaluated by FACS analysis; open curves represent isotype controls. Data are from a representative experiment, which was done 3 times with similar results. (b) Chemotaxis of LLC cancer cells to PMV. LLC cells were resuspended in 0.5% BSA + RPMI and placed in the upper chambers of transwells. Transwell membranes were covered with laminin. The lower chambers contained medium with 10 (low dose) or 50 (high dose) μg/mL of PMV. After 24 hr the cells on the lower portion of the transwell membrane were stained and counted under an inverted microscope. Data from 3 separate experiments were pooled together and means ± SD are shown. (c) PMV increase proliferation of LLC cells. LLC cells were cultured in 0.5% BSA for 72 hr in the absence (left bar) or presence of PMV (low dose, 10 μg/mL) (right bar). The experiment was done 3 times in quadruplicate with similar results. *p < 0.00001 as compared to control (0.5% BSA). (d) Phosphorylation of MAPK p42/44 and AKT by PMV. Phosphorylation of MAPK p42/44 and AKT in LLC cells. In each panel: lane 1, quiescent LLC cells (control); lane 2, LLC cells stimulated for 5 min by low dose of PMV (10 μg/mL); lane 3, LLC cells stimulated for 5 min by high dose of PMV (30 μg/mL). The experiment was done twice with similar results. A representative study is shown. (e,f) Metastasis of Lewis lung carcinoma (LLC) cells to bone marrow. C57Bl6 mice were injected i.v. with 1 × 105 LLC cells. (e) The morphologic analysis (hematoxylin-eosin staining × 670) of a representative bone marrow section of mice receiving LLC cells not covered with PMV. (f) A representative picture (hematoxylin-eosin staining × 670) of a bone marrow slide from mice that were injected with PMV-covered LLC cells. Arrows indicate LLC cells.

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Stimulation of A549 cells with PMV increases expression of angiogenic factors

As described above, we found that PMV stimulated, besides proliferation, various aspects of the metastatic behavior (e.g., chemotaxis, chemoinvasion, adhesion, MMP expression) of lung cancer cells. Hence, we investigated whether PMV could also affect the angiogenic potential of lung tumors. We stimulated A549 cells with both surface-derived PMV and exosomes and evaluated changes in expression of mRNA for VEGF, HGF and IL-8, as well as MMPs. We found that A549 cells stimulated by PMV exhibited about 35-fold more mRNA for IL-8 than unstimulated controls (Table II), and those cells stimulated by microvesicles enriched in exosomes about 17-fold more of it. Expression of mRNA for VEGF and HGF was 3–4-fold higher (Table I) in PMV-stimulated A549 cells. Thus it appears that PMV also increase the expression of angiogenic factors necessary for the metastatic process.

Table II. Real-Time RT-PCR Analysis of Changes in mRNA Expression for Angiogenic Genes in A549 Cells Stimulated for 4 hr by PMV (30 μg/mL)
 PMV fold increaseMV enriched in exosomes fold increase
  1. PMV, platelet-derived microvesicles; MV, microvesicles.

VEGF3 ± 0.54 ± 1
IL-835 ± 917 ± 4
HGF4 ± 13 ± 1

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

It has been reported that platelets may exacerbate tumor progression/metastasis and angiogenesis.43, 44, 45, 46 Induction of thrombocytopenia reduces experimental metastases, which can be increased, on the other hand, by platelet transfusions.46 In this work, we hypothesized that these platelet-related effects are mediated by microvesicles or exosomes released from activated platelets, and hence we examined their influence on biologic processes related to the progression, metastasis and angiogenesis of lung tumors. In our experiments, we employed doses of PMV that we considered to be clinically relevant.5, 53

Our major finding is that PMV derived from activated platelets interact with lung cancer cells and enhance their metastatic and angiogenic potential. PMV chemoattracted 5 out of the 6 human and murine lung cancer cell lines studied, with the strongest effects shown by the highly metastatic human A549 cell line and the murine LCC cells. Moreover, we found that interaction of PMV with most of the lung cancer cell lines studied resulted in the activation of MAPK p42/44 and AKT, signaling pathways participating in proliferative responses. We demonstrated increased proliferation of A549 and LCC cells consistent with our previous work showing enhancement by PMV of the proliferation of normal and malignant human hematopoietic cells, which was also accompanied by the phosphorylation of MAPK p42/44 and PI-3K–AKT,12 indicating that PMV are able to interact directly with target cells and act as a signaling molecule.4 It has been reported that PMV activate other signaling pathways as well, e.g., in U-937 cells PMV stimulated PKC, p38 and JNK1.3, 19, 24 Furthermore, in addition to the activation of signaling pathways, we found that PMV upregulated the expression of mRNA for cyclin D2 in A549 cells. Overexpression of cyclin D2 has been associated with increased invasiveness in vivo and progression of various tumors such as human squamous carcinoma cells, testicular germ tumors and breast cancer cells.48 Hence we can assume that activation of cyclin D2 by PMV could likewise induce a more invasive phenotype in lung cancer cells.

Next, we demonstrated that PMV upregulated mRNA expression of MT1-MMP, in 3 of 5 lung cancer cell lines, with the strongest effect demonstrated on HTB 177 cells and translated to a high MT1-MMP level and enhanced chemoinvasion. The MT-MMPs are highly expressed in almost all types of human cancer.29, 30 MT1-MMP has been localized predominantly to specialized membrane extensions, and this localization appears to be essential for cancer cell invasion.30 However, MT1-MMP regulation of cell invasion is not related exclusively to its catalytic activity against extracellular matrix components or activation of proMMP-2. Recent evidence indicates that MT1-MMP cleaves a multifunctional adhesion molecule CD44 and induces cell migration.49 MT1-MMP also cleaves cell surface transglutaminase, an integrin-binding adhesion co-receptor that inhibits cell migration on fibronectin but enhances it on collagen.50 Thus, by means of their specific cell-surface localization and substrate profile, MT-MMPs can modulate the necessary molecular mechanisms that control the migratory and invasive phenotype of tumor cells. Moreover, a direct role for MT1-MMP in tumor cell proliferation was recently postulated.51 MMP-9 and MMP-2 were found to be expressed only by the highly metastatic A549 cells, and PMV stimulated the mRNA expression of MMP-9 and the activation of proMMP-2 in this cell line, which translated to increased chemoinvasion across Matrigel.

PMV enhancement of the metastatic potential of lung cancer cells is further demonstrated by our findings that PMV increase adhesion of tumor cells to endothelial cells and fibrinogen by transfer to their surface of various platelet-derived integrins such as CD41. Accompanied by increased MMP activity, this greater adhesiveness could promote transendothelial migration of these cells into the tissue. Further supporting this is our previous finding that hematopoietic stem cells covered with PMV home to the bone marrow and engraft faster after transplantation than stem cells that were not covered with PMV.11

Another important aspect of tumor progression is tumor vascularization. We demonstrated that stimulation of lung cancer cells by PMV upregulates the expression of several genes involved in tumor vascularization, namely VEGF, IL-8 and HGF, in addition to MMPs. Thus, our in vitro data show that PMV exert pleiotropic effects on many biologic processes central to tumor progression, metastasis and angiogenesis.

Lastly, we present in vivo evidence showing that murine LLC cells covered with PMV acquired higher metastatic potential than cells not so covered. Not only were significantly more metastatic foci found in the lungs of mice injected with PMV-covered LCC cells, but also there were significantly more LCC cells detected in the bone marrow cavities of these mice. As PMV have been shown to transfer G-protein-coupled 7-transmembrane-span receptors such as CXCR4 onto the surface of target cells,11, 12, 14 it is reasonable to suggest that LCC cells pre-incubated with PMV acquired CXCR4 on their surface and responded to the SDF-1-rich environment of the bone marrow. Previously, we reported that all 5 human lung cancer cell lines studied here do not express the CXCR4 receptor33; however, as shown in other models, cells can be rendered CXCR4-positive by PMV, and CXCR4 transferred by PMV may be functional as a coreceptor for HIV entry14 or tether PMV+ cells to SDF-1 exposed on endothelium.12

Although it is apparent that PMV contain components that alone or in combination are responsible for the various effects observed, the particular components responsible remain to be elucidated. The influence of PMV on chemotaxis, for example, may be attributed to the presence of S1P or AA.13, 20, 52 Unsaturated acids, however, have been found to induce apoptosis and inhibit proliferation in several other experimental systems.13, 52 Our previous studies have demonstrated that the biologic activity of PMV is associated with high-molecular-weight proteins and is only partly affected by protein digest and/or heat inactivation.12 We suggested that it could be related partly to high-molecular-weight, relatively temperature-stable proteins and partly to bioactive lipids conjugated with high-molecular-weight proteins.

In conclusion, based on our studies, we postulate that human PMV are important but still underappreciated mediators of intercellular cross-talk between platelets and cancer cells and modulate their functions. The data from this work may explain, at least in part, the exacerbating effects of high platelet counts on cancer progression43, 44, 46 by the action of PMV and exosomes derived from them. Further supporting our data is a recent report showing that the increased level of PMV circulating in PB is a strong predictor of metastasis in patients with gastric cancer.53 More studies, however, are needed to identify the specific PMV components that exert these biologic effects, as a better understanding of the role PMV play in tumor progression and metastasis could help us to develop novel therapeutic approaches.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
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