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[Cancer Research 62, 6312-6317, November 1, 2002]
© 2002 American Association for Cancer Research

Tumor Biology

Extracellular Membrane Vesicles from Tumor Cells Promote Angiogenesis via Sphingomyelin1

Chan Woo Kim, Hwan Myung Lee, Tae Hoon Lee, Chulhun Kang, Hynda K. Kleinman and Yong Song Gho2

Department of Oncology, Graduate School of East-West Medical Science, Kyung Hee University, Yong In, 449-701, Korea [C. W. K., H. M. L., T. H. L., C. K., Y. S. G.], and Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, Maryland 20892 [H. K. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Actively growing tumor cells shed membrane vesicles into the extracellular milieu both in vivo and in vitro. Extracellular membrane vesicles from tumor cells contain most surface antigens and proteases present on these cells. They facilitate the escape of tumors from immune surveillance and promote tumor cell invasion. Here, we demonstrate that tumor membrane vesicles stimulate an additional important activity for tumor growth and metastasis by promoting endothelial cell migration, invasion, and tube formation, and inducing in vivo neovascularization. Our data show that tumor vesicles are one of the multiple effectors involved in tumor-induced angiogenesis. Heat-treated vesicles and lipid extracts from the vesicles also induce endothelial cell migration and in vivo angiogenesis. We identify sphingomyelin as the active component for vesicle-induced endothelial cell migration, tube formation, and neovascularization. Together with previously reported results, our data demonstrate that shed tumor vesicles play multiple roles in tumor growth and metastasis by promoting angiogenesis, tumor invasion, and immune escape.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activated cells, including endothelial cells, platelets, and monocytes, shed fragments of their plasma membrane into the extracellular milieu under specific physiological conditions (1, 2, 3) . The process of shedding is elevated in actively growing tumor cells that continually shed membrane vesicles in vitro and in vivo (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . Although several hypotheses have been suggested, the exact mechanisms involved in shedding extracellular membrane vesicles are not clear (4 , 5) . EMVTCs3 are derived from selected areas of the plasma membrane (6, 7, 8) and appear to be enriched in most surface antigens and proteases present on tumor cells (9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . When compared with the plasma membrane, EMVTCs are composed of a lower content of phospholipids but an increased amount of cholesterol and sphingomyelin, resulting in a more rigid structure (7 , 8) . Several studies have suggested that EMVTCs are involved in tumor growth and metastasis by playing relevant roles in the escape of tumors from immune surveillance and in promoting tumor cell invasion (9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . Tumor surface antigens and the immune-suppressing cytokine, transforming growth factor-ß, in EMVTCs provide powerful factors that protect tumors from immune attack (9, 10, 11, 12) . The levels of membrane vesicles in the sera of cancer patients are significantly higher than that of healthy donors (13 , 14) . Several proteases, including MMPs and plasminogen activator, are enriched in EMVTCs (13, 14, 15, 16, 17, 18) . These proteolytic enzymes and integrins in EMVTCs are thought to play important roles in tumor cell invasion and metastasis. Both the amounts and the vesicle-associated proteolytic activities of EMVTCs are positively correlated with malignancy in vivo and with the invasiveness of tumor cells in vitro (14 , 15) .

In addition to immune escape and tumor invasion, angiogenesis, the formation of new capillaries from the preexisting blood vessels, is critical for tumor growth and metastasis (19 , 20) . Actively growing tumor cells need increased blood vessels to take up nutrients and clean up wastes for their survival. Furthermore, such vessels could provide pathways for metastasis. Here we show that EMVTCs have angiogenic activity and that sphingomyelin may be an active component involved in EMVTC-induced neovascularization.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
Immortalized HMEC-1 were maintained in RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin, 3 ng/ml human bFGF (R&D Systems, Minneapolis, MN), and 5 units/ml heparin (Sigma-Aldrich, St. Louis, MO). Primary HUVECs were prepared and cultured as described previously (21) . HT1080 human fibrosarcoma and DU-145 human prostate carcinoma cells were maintained in RPMI 1640 supplemented with 10% FBS and penicillin-streptomycin.

EMVTC Preparation.
EMVTCs were prepared as described previously (16) . Briefly, confluent HT1080 and DU-145 cells were washed and grown in serum-free RPMI 1640. After a 24-h incubation, conditioned medium was removed and centrifuged at 500 x g for 10 min and then at 800 x g for 15 min. The supernatant was centrifuged at 100,000 x g for 1 h. The resulting pelleted EMVTCs were resuspended in PBS, and then the protein concentration was determined (22) . EMVTCs were aliquoted and stored at -20°C until use. For heat treatment, EMVTCs were boiled for 10 min.

For further purification of EMVTCs, centrifugation of EMVTCs on a discontinuous sucrose gradient was carried out at 4°C (23) . Briefly, 1.5 ml of 1.4 M, 1.0 M, and 0.6 M sucrose in 20 mM HEPES/NaOH (pH 7.2) was added to a Sorvall AH-650 tube. A solution of EMVTCs in PBS (0.5 ml) was overlaid on top of the discontinuous sucrose gradient, and the sample was centrifuged at 150,000 x g for 2 h. Fractions of the gradient (0.8 ml each) were diluted in 4.2 ml of PBS and were centrifuged at 100,000 x g for 2 h,, and each individual pellet was resuspended in PBS. Then, the protein concentration was determined. The EMVTCs were recovered from the 1.0–1.4-M sucrose interface as reported previously (12) .

Migration and Invasion Assay.
Endothelial migration and invasion assays were carried out in 48-well microchemotaxis chambers (Neuro Probe Inc., Cabin John, MD) as described previously (21) . Polyester membranes with 12-µm pores were coated with a 0.1% gelatin (Sigma-Aldrich) in deionized water and a 0.5 mg/ml Matrigel (Collaborative Biomedical Products, Bedford, MA) in double distilled water for the migration and invasion assays, respectively. Human endothelial cells were resuspended in RPMI 1640 containing 0.1% BSA. The bottom chamber was loaded with 30,000 cells, and the membrane was laid over the cells. The chamber was inverted and incubated for 2 h. Upper wells were then loaded with RPMI 1640 containing 0.1% BSA and test samples. The chamber was then reincubated for 2 h and 4 h for migration and invasion assays, respectively. The membrane filters were fixed and stained using Diff-Quick (Baxter Healthcare Corp., McGraw Park, IL). The number of cells that migrated and invaded through the filter was determined by counting two regions of each well under a microscope. All of the samples used in the experiments with lipids contained 0.1% DMSO, and the presence of DMSO did not affect endothelial cell migration. Protease inhibitor cocktail, comprising the mixture of Complete, Mini (Roche Molecular Biochemicals, Mannheim, Germany) and MMP-2/MMP-9 inhibitor II (40 ng/ml; Calbiochem, La Jolla, CA) was used in the invasion assay. Aprotinin (3 µg/ml) and MMP-2/MMP-9 inhibitor II (20 ng/ml) were also used in additional invasion assays. Experiments were carried out in triplicate and repeated at least twice.

Capillary-like Tube Formation Assay.
To investigate the in vitro angiogenic activity, the tube formation assay was performed as described previously (21) . Briefly, HMEC-1 (24,000 cells) in 0.4 ml of serum-free RPMI 1640 with various concentrations of EMVTCs were added to each well, coated with 1:1 mixture of RPMI 1640 and Matrigel. After a 3-h incubation, two randomly chosen fields (x10) from each sample were photographed, and total tube areas were analyzed by the Scion Image program. Analyses of all of the test samples were performed in triplicate.

CAM Assay.
To investigate the in vivo angiogenic activity, the modified chick CAM assay was carried out as described previously (21) . Briefly, 18 µl of test samples in rat tail type I collagen (Collaborative Biomedical Products) was applied onto Thermonox disks (Nunc, Naperville, IL) and polymerized by warming. The discs were loaded onto the CAM of 10-day-old embryos. After a 72-h incubation, the area around the loaded disk was photographed with a Nikon digital camera, and the number of newly formed vessels was counted by two observers (C.W.K. and T.H.L.) in a double-blind manner. Assays for each test sample were carried out using 10–13 eggs.

Lipid Extraction and TLC.
We extracted total lipids from EMVTCs using chloroform/methanol (24) . Total lipids were separated by TLC with chloroform/methanol/H2O (7:3:1, v/v/v). For preparation of sphingomyelin from EMVTCs, extracted lipids were separated by TLC and the area with an Rf value identical to that of bovine brain sphingomyelin (Sigma-Aldrich) was scraped. After the addition of chloroform/methanol (1:2, v/v), the mixture was centrifuged. Sphingomyelin was recovered by evaporation of the lower organic phase with a stream of nitrogen and was dissolved in DMSO. Commercially obtained sphingomyelin was also purified by this TLC method.

Sphingomyelinase Assay.
S1P (Biomol, Plymouth Meeting, PA), bovine sphingomyelin, total lipid extracts, and purified sphingomyelin from EMVTCs were treated with sphingomyelinase (100 units/ml; Sigma-Aldrich) in 250 mM Tris-HCl (pH 7.4), containing 0.1% Triton X-100 and 20 mM MgCl2. After a 3-h incubation at 37°C, lipids were extracted and fractionated by TLC as described above.

Statistical Analysis.
All of the values are expressed as mean ± SD. Ps were calculated from Student’s t test, based on comparisons with appropriate control samples tested at the same time.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
EMVTCs Stimulate Endothelial Cell Migration and Invasion.
Because endothelial cell migration and invasion through extracellular matrix are essential for neovascularization, we first investigated the effects of EMVTCs on endothelial cell migration and invasion. One million HT1080 cells shed ~2 and 6 µg of EMVTCs per day in the absence and presence of serum, respectively. In all of our experiments, we used EMVTCs prepared from serum-free conditions. EMVTCs significantly induced up to a 2.7-fold increase in the migration of HMEC-1s in a dose-dependent manner over migration in the presence of medium alone (Fig. 1A)Citation . Endothelial cell migration, stimulated by 5 µg/ml EMVTCs, was similar to levels observed for the positive control bFGF (5 ng/ml). EMVTCs also stimulated the migration of HUVECs with a response comparable with that observed with HMEC-1 (data not shown). EMVTCs, purified by sucrose gradient sedimentation, also induced endothelial cell migration, and the migratory activity was similar to that of EMVTCs prepared by ultracentrifugation (Fig. 1BCitation ; P > 0.5). These results clearly indicate that EMVTCs have the biological activities that can induce endothelial cell migration. Heat-treated EMVTCs induced endothelial cell migration at a level similar to that of untreated EMVTCs, which suggests that the major active factor(s) may not be a protein (Fig. 1CCitation ; P > 0.1). Serum-free tumor-conditioned medium induced a 3.4-fold increase in the migration of HMEC-1 cells (Fig. 1D)Citation . Conditioned medium, after the removal of EMVTCs, still induced endothelial cell migration, but showed significantly reduced migratory activity compared with that of conditioned medium itself (P < 0.00001). These results suggest that both EMVTCs and factors not associated with EMVTCs in tumor-conditioned medium are involved in stimulating endothelial cell migration.



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  		Fig. 1. EMVTCs and heat-treated EMVTCs stimulate endothelial cell migration. In A, EMVTCs significantly induced migration of HMEC-1s over migration in the presence of medium alone. Positive control, bFGF, showed a 2.0-fold increase in migration. HUVECs also showed similar responses to EMVTCs (not shown). In B, EMVTCs, purified by sucrose gradient sedimentation ({blacksquare}), also induced endothelial cell migration, and its migratory activity was similar to that of EMVTCs prepared by ultracentrifugation ({square}). In C, heat-treated EMVTCs ({blacksquare}) showed similar migratory activity compared with that of untreated EMVTCs ({square}). In D, EMVTCs are the active factors in tumor-conditioned medium involved in stimulation of endothelial cell migration. Serum-free tumor-conditioned medium after the removal of EMVTCs ({blacksquare}) showed significantly reduced migratory activity compared with that of tumor-conditioned medium ({square}) but still induced endothelial cell migration over that observed with control medium alone ().

 
We next investigated the effect of EMVTCs on the invasion of endothelial cells. EMVTCs significantly induced endothelial cell invasion about 2-fold in a dose-dependent manner (Fig. 2A)Citation . Protease inhibitor cocktail, comprising the mixture of Complete, Mini and MMP-2/MMP-9 inhibitor II, blocked EMVTC-induced endothelial cell invasion (Fig. 2B)Citation , but it did not affect EMVTC-induced endothelial cell migration (data not shown). Complete, Mini inhibits a broad spectrum of serine and cysteine proteases as well as calpains. Because EMVTCs are enriched with plasminogen activator, MMP-2, and MMP-9, which are essential for cell invasion through extracellular matrix (13, 14, 15, 16, 17, 18) , we investigated the involvement of these proteases in EMVTC-induced endothelial cell invasion. Aprotinin (3 µg/ml) and MMP-2/MMP-9 inhibitor II (20 ng/ml) blocked EMVTC-induced endothelial cell invasion (Fig. 2CCitation ; P < 0.0001). A mixture of these inhibitors further blocked EMVTC-induced endothelial cell invasion, and the inhibitory activity was stronger than that of aprotinin or MMP-2/MMP-9 inhibitor alone (P < 0.001). The presence of the tissue inhibitor of metalloproteinase-1 (1 µg/ml), the tissue inhibitor of metalloproteinase-2 (2 µg/ml), the plasminogen activator inhibitor-1 (10 µg/ml), or the {alpha}2-antiplasmin (10 µg/ml) efficiently reduced EMVTC-induced endothelial cell invasion (Fig. 2, D and ECitation ; P < 0.00001). All of the protease inhibitors also blocked the basal invasion observed in the absence of EMVTCs, which indicated the expected role of endogenous proteases. These results indicate that both the MMPs and the plasminogen activator present in EMVTCs may be involved in the stimulation of endothelial invasion. Heat treatment decreased the ability of EMVTCs to stimulate endothelial cell invasion (P < 0.00001), but heat-treated EMVTCs still induced the invasion of endothelial cells more so than the invasion in the presence of medium alone (Fig. 2FCitation ; P < 0.00001); this suggests that residual proteolytic activities may still be present in the boiled sample and/or that a nonprotein factor(s), along with proteases in EMVTCs, may contribute to endothelial cell invasion.



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  		Fig. 2. EMVTCs stimulate endothelial cell invasion. In A, EMVTCs significantly induced invasion of endothelial cells over invasion in the presence of medium alone. Positive control, bFGF, showed a 1.5-fold increase in invasion. In B, protease inhibitor cocktail blocked EMVTC-induced endothelial cell invasion. However, this treatment did not affect EMVTC-induced endothelial cell migration (not shown). In C, aprotinin and MMP-2/MMP-9 inhibitor II blocked EMVTC-induced ({square}) and basal invasion of endothelial cells ({blacksquare}). In D and E, tissue inhibitor of metalloproteinase-1 and -2, plasminogen activator inhibitor-1, and {alpha}2-antiplasmin efficiently reduced both EMVTC-induced ({square}) and basal invasion of endothelial cells ({blacksquare}). TIMP, tissue inhibitor of metalloproteinase; PAI, plasminogen activator inhibitor; Antiplasmin, {alpha}2-antiplasmin. In F, heat-treated EMVTCs ({blacksquare}), compared with untreated EMVTCs ({square}), showed a decrease in the ability to stimulate endothelial cell invasion.

 
EMVTCs Have Angiogenic Activity.
We next examined the ability of EMVTCs to promote the formation of capillary-like structures of HMEC-1 cells on Matrigel. This assay measures activities involved in some of the steps in neovascularization, including migration and the differentiation of endothelial cells (25) . The presence of EMVTCs at 10 µg/ml, 20 µg/ml, and 40 µg/ml showed a significant 1.4-, 1.8-, and 2.0–fold increase, respectively, in the tube area over control containing medium alone (Fig. 3ACitation ; P < 0.01). Tube formation stimulated by 20 µg/ml EMVTCs was similar to levels observed for the positive control bFGF (5 ng/ml; 1.8-fold increase). Heat-treated EMVTCs promoted the formation of capillary-like structures at a level similar to that of untreated EMVTCs (P > 0.5). EMVTCs did not act as a mitogen for endothelial cells (data not shown).



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  		Fig. 3. EMVTCs have angiogenic activity in vitro and in vivo. In A, EMVTCs ({square}) and heat-treated EMVTCs ({blacksquare}) stimulated the formation of a capillary-like structures by endothelial cells on Matrigel over control medium alone (). In B and C, EMVTCs and heat-treated EMVTCs stimulated neovascularization in the chick CAM assay. Representative photographs of disks and surrounding CAMs (B) and quantification of newly formed blood vessels (C). , vehicle; {square}, EMVTCs, {blacksquare}, heat-treated EMVTCs.

 
We next examined whether EMVTCs stimulated in vivo angiogenesis using the chick CAM assay. EMVTCs significantly induced neovascularization from preexisting blood vessels (Fig. 3, B and C)Citation . The presence of 1 and 3 µg of EMVTCs per egg caused 2.9-fold and 3.6-fold increases, respectively, in the number of newly formed blood vessels compared with that of PBS alone (P < 0.00001). Heat-treated EMVTCs also stimulated blood vessel formation in the CAM assay with angiogenic activity comparable with that of the untreated EMVTCs (P > 0.1). These results clearly indicate that EMVTCs has angiogenic activity and that a component(s) resistant to heat treatment may be involved in its activity.

Sphingomyelin in EMVTCs Stimulates Endothelial Cell Migration and Has Angiogenic Activity.
Because the migratory and angiogenic activities of EMVTCs were heat resistant, we examined the angiogenic activities of lipids extracted from EMVTCs. The lipids in the organic phase (after the extraction of EMVTCs by the Folch method) induced endothelial cell migration in a dose-dependent manner (P < 0.00001) with migratory activity similar to that of EMVTCs (Fig. 4ACitation ; P > 0.5). TLC analysis showed that both EMVTCs and EMVTCs that were purified by sucrose gradient sedimentation contained several abundant lipids, including sphingomyelin and other phospholipids, and that their lipid composition was similar to each other (data not shown). We examined the angiogenic activities of the lipids present in EMVTCs. Sphingomyelin from bovine brain stimulated the migration of endothelial cells (Fig. 4BCitation ; P < 0.0001), with maximal migratory activity at 0.5 µg/ml (a 4.1-fold increase). Heat-treated sphingomyelin also stimulated the migration of endothelial cells. The purity of the commercially obtained bovine sphingomyelin used in this study is ~99%. Sphingomyelin, further purified by TLC, also stimulated endothelial cell migration, and the activity was comparable with that of the unpurified material (data not shown). The other major phospholipids, such as phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and phosphatidylserine, that were present in EMVTCs showed little effect on endothelial migration (data not shown). Sphingomyelin stimulated the invasion of endothelial cells, and gelatin zymography showed that the presence of sphingomyelin at concentrations of up to 1 µg/ml did not up-regulate MMP activity in HUVECs (data not shown). Sphingomyelin also promoted capillary tube-like formation of endothelial cells on Matrigel (Fig. 4CCitation ; P < 0.0001). The positive control, bFGF (5 ng/ml), showed a 2.5-fold increase in tube formation, whereas the negative control, the medium alone, showed very low activity.



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  		Fig. 4. Sphingomyelin in EMVTCs has angiogenic activity. In A, lipids extracted from EMVTCs ({blacksquare}) induced endothelial cell migration, and the migratory activity is similar to that of EMVTCs ({square}). The EMVTC-derived lipid concentrations used here are unknown but equivalent to what is isolated from the indicated amount of EMVTCs. In B, bovine sphingomyelin ({square}) and heat-treated sphingomyelin ({blacksquare}) stimulated migration of endothelial cells. Sphingomyelin also stimulated the invasion of endothelial cells (not shown). In C, sphingomyelin ({blacksquare}) stimulated the formation of capillary-like structures by endothelial cells on Matrigel over that observed with control medium alone ({square}). In D, sphingomyelin, purified from EMVTCs (), stimulated the migration of endothelial cells and this activity was comparable with that of EMVTCs ({square}) and of heat-treated EMVTCs ({blacksquare}). The EMVTC-derived sphingomyelin concentrations used here are unknown but are equivalent to what is isolated from the indicated amount of EMVTCs. In E, TLC of sphingomyelinase (SMase)-treated crude lipid extracts from EMVTCs, bovine sphingomyelin, and S1P. Crude Lipids, crude lipid extracts from EMVTCs, SM, bovine sphingomyelin. Treatment of purified sphingomyelin from EMVTCs with sphingomyelinase also caused a disappearance of sphingomyelin (not shown). Arrows, sphingomyelin. In F, sphingomyelinase-treated bovine sphingomyelin (0.5 µg/ml) and crude lipids from EMVTCs ({blacksquare}) lost their migratory activity when compared with untreated material ({square}). However, sphingomyelinase treatment did not affect the migratory activity of S1P (0.4 µg/ml). The EMVTC-derived lipid concentrations used here are unknown but equivalent to 25 µg/ml EMVTCs used for lipid extraction.

 
We next purified the sphingomyelin present in EMVTCs by TLC and examined its angiogenic activity. Sphingomyelin, purified from EMVTCs, stimulated the migration of HUVECs (P < 0.001), and this activity was comparable with that of EMVTCs and of heat-treated EMVTCs (Fig. 4DCitation ; P > 0.1). TLC analysis showed that treatment of crude lipid extracts and purified sphingomyelin from EMVTCs with sphingomyelinase caused a disappearance of the spot with an Rf value identical to that of sphingomyelin (Fig. 4E)Citation . The other lipids in EMVTCs were not affected by this enzyme. Furthermore, treatment of bovine sphingomyelin and crude lipid extracts from EMVTCs with sphingomyelinase almost completely abolished their migratory activity (Fig. 4FCitation ; P < 0.01). To investigate the possibility that S1P, the most potent endothelial cell chemotactic and angiogenic lipid, might be involved in EMVTC-mediated endothelial cell migration, we examined the effect of sphingomyelinase treatment on the migratory activity of S1P. Sphingomyelinase treatment did not degrade S1P as observed by TLC analysis (Fig. 4E)Citation , nor did it affect the migratory activity of S1P (Fig. 4FCitation ; P > 0.5), which suggested that S1P may not be the active component involved in EMVTC-mediated endothelial cell migration. We further confirmed that the active component of EMVTCs was sphingomyelin by the CAM assay (Table 1)Citation . Lipid extracts and purified sphingomyelin from 3 µg of EMVTCs caused 5.2-fold and 5.3-fold increases, respectively, in the number of newly formed blood vessels in the chick CAM assay when compared with the number of newly formed blood vessels when using PBS alone (P < 0.00001). These levels of activities were similar to that of EMVTCs (P > 0.1). TLC analysis showed that 3 µg of EMVTCs contained about 0.3 µg of sphingomyelin. Bovine sphingomyelin (0.3 µg) also induced a 5.8-fold increase in neovascularization over that induced by PBS control (P < 0.00001), and this activity is almost identical to that of EMVTCs itself, lipid extracts, and purified sphingomyelin from EMVTCs (P > 0.1). These results suggest that sphingomyelin may be an active component involved in EMVTC-induced angiogenic activity.


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  		Table 1 Angiogenic activity of lipid extracts, purified sphingomyelin from EMVTC, and bovine sphingomyelin in the chick CAM assay

 
We used EMVTCs that were shed by human fibrosarcoma cells in all of the assays described above. We prepared EMVTCs from human prostate adenocarcinoma cells (DU-145) to demonstrate that the angiogenic activity is present in the EMVTCs that come from other tumor cell types. These EMVTCs also induced endothelial cell migration, and heat-treated EMVTCs showed similar migratory activity compared with that of untreated EMVTCs (data not shown). TLC analysis of extracted lipids showed that sphingomyelin and other phospholipids were present in EMVTCs from DU-145 cells (data not shown). These data suggest that the EMVTCs from different tumor cell types have a similar capacity for angiogenesis.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this report are that EMVTCs have angiogenic activity and that sphingomyelin may be the active component involved in this activity. EMVTCs prepared from human fibrosarcoma cells significantly induced endothelial cell migration, morphogenesis, and in vivo neovascularization, and heat treatment did not affect these activities. EMVTCs did not act as a mitogen for endothelial cells. In addition to factors not associated with membrane vesicles, EMVTCs are also involved in tumor-induced angiogenesis. EMVTCs stimulate endothelial cell invasion. Proteases, including MMPs and plasminogen activator that are enriched in membrane vesicles, are involved in EMVTC-induced endothelial cell invasion. Extracted lipids from EMVTCs also induced endothelial cell migration and in vivo angiogenesis. Furthermore, sphingomyelin, one of the major lipid components in EMVTCs, showed almost identical migratory and angiogenic activities as EMVTCs. Heat treatment did not affect the migratory activity of sphingomyelin. Sphingomyelinase-treated sphingomyelin and crude lipid extracts from EMVTCs lost their migration-promoting activity, whereas the migratory activity of S1P was not affected by sphingomyelinase. Our data demonstrate that EMVTCs from another human cancer cell line, DU-145, also stimulated endothelial cell migration and contained sphingomyelin.

It is surprising that lipid components, such as sphingomyelin, are involved in EMVTC-mediated neovascularization. Sphingomyelins are one of the major membrane phospholipids, and most are localized on the outer leaflet of the mammalian plasma cell membrane (26) . The sphingomyelin levels of highly metastatic cancer cells are significantly higher than that of less metastatic variants (27) . EMVTCs are composed of increased amounts of sphingomyelin relative to that of the plasma membrane (7 , 8) . Sphingolipids play diverse roles in cell proliferation, differentiation, apoptosis, and migration (28, 29, 30, 31) . Sphingolipids, including S1P, sphingosylphosphorylcholin, sphingosine, and gangliosides, are also involved in angiogenesis via their intrinsic angiogenic activities or promotion of the angiogenic response of microvessels stimulated by angiogenic factors (32, 33, 34, 35, 36) . In our studies, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and phosphatidylserine, which are present in EMVTCs, showed little effect on endothelial cell migration. It has been reported that sphingomyelin is less potent for cell migration than S1P is (32) . However, our results clearly indicate that sphingomyelin itself, as well as the sphingomyelin present in EMVTCs, has angiogenic activity. Furthermore, sphingomyelinase treatment did not either degrade S1P nor affect its migratory activity, whereas sphingomyelinase-treated sphingomyelin and sphingomyelinase-treated crude lipid extracts from EMVTCs lost almost all of their migration-promoting activity with this treatment. These results suggest that S1P may not be the active component involved in the EMVTC-mediated angiogenesis. Although we could not completely exclude the possibility that other lipid components are involved in EMVTC-induced angiogenesis, our results indicate that sphingomyelin may be a major active component. It is important to note that the serum level of shed vesicles in cancer patients (80 µg/ml) relates closely to the amounts used here in our assays (6–50 µg/ml) and that have been shown to be active for angiogenesis. The level of extracellular membrane vesicles observed in cancer patients is approximately five times that observed in normal patients, as expected because normal cells shed much less material than do cancer cells (14 , 37) . Recently, it was demonstrated that membrane vesicles that were shed by activated endothelial cells were enriched with MMPs and promoted endothelial cell invasion and tube formation on Matrigel (37) . Although it has not been tested, one would expect that the shed vesicles from normal cells may also have angiogenic activity based on the lipid composition.

What might be the significance of the angiogenic activities of EMVTCs in tumor growth and metastasis? Several studies have suggested that EMVTCs are involved in both immune escape and tumor invasion via EMVTC-associated tumor cell antigens, immune-suppressing cytokines, integrins, and proteases (9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . Actively growing tumor cells shed EMVTCs via a vital process, and the rate of shedding is increased in malignant tumors. Evidence suggests that the serum amounts of EMVTCs in cancer patients are significantly elevated, and that both the amounts and the vesicle-associated proteolytic activities of EMVTCs are positively correlated with malignancy and the invasiveness of tumor cells in vitro (13, 14, 15) . In addition to tumor cell invasion and the escape of tumors from immune surveillance, angiogenesis is essential for tumor growth and metastasis. It is worthwhile to note that even a single molecule, such as bFGF, and several soluble cell-adhesion molecules, including E-selectin, vascular cell adhesion molecule, and intercellular adhesion molecule-1, perform dual functions: angiogenesis and escape from immune surveillance (21 , 38, 39, 40) . EMVTCs, composed of multiple proteins and lipids, may play an additional role in angiogenesis as well as in immune escape and tumor invasion. Together with previously reported results, our data suggest that EMVTCs may play a major role in tumor cell survival in the host. EMVTCs are fully equipped with the tumor antigens, proteases, and angiogenic molecules needed for immune escape, tumor invasion, and neovascularization and, thus, play a triple role in tumor growth and metastasis. Although additional work on the physiological significance of EMVTC-induced angiogenesis in tumor growth and metastasis is required, our findings suggest that blocking the shedding of EMVTCs could be a worthwhile approach for cancer therapy.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-B-20800-0077). C. W. K., H. M. L., and T. H. L. were supported by the BK21 program of the Ministry of Education. Back

2 To whom requests for reprints should be addressed, at Department of Oncology, Graduate School of East-West Medical Science, Kyung Hee University, Yong In 449-701, Korea. Phone: 82-31-201-2178; Fax: 82-31-204-8119; E-mail: ysgho{at}khu.ac.kr Back

3 The abbreviations used are: EMVTC, extracellular membrane vesicle(s) from tumor cell; MMP, matrix metalloproteinase; HMEC-1, immortalized human microvascular endothelial cell(s); FBS, fetal bovine serum; bFGF, basic fibroblast growth factor; HUVEC, human umbilical vein endothelial cell; CAM, chorioallantoic membrane; TLC, thin-layer chromatography; S1P, sphingosine 1-phosphate. Back

Received 3/ 5/02. Accepted 8/30/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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