Abstract
Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P < 0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P < 0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P < 0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.
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References
Folkman J (2002) Role of angiogenesis in tumor growth and metastasis. Semin Oncol 29:15–18
Aguayo A, Kantarjian H, Manshouri T, Gidel C, Estey E, Thomas D, Koller C, Estrov Z, O’Brien S, Keating M, Freireich E, Albitar M (2000) Angiogenesis in acute and chronic leukemias and myelodysplastic syndromes. Blood 96:2240–2245
Lundberg LG, Lerner R, Sundelin P, Rogers R, Folkman J, Palmblad J (2000) Bone marrow in polycythemia vera, chronic myelocytic leukemia, and myelofibrosis has an increased vascularity. Am J Pathol 157:15–19
Zhelyazkova AG, Tonchev AB, Kolova P, Ivanova L, Gercheva L (2008) Prognostic significance of hepatocyte growth factor and microvessel bone marrow density in patients with chronic myeloid leukaemia. Scand J Clin Lab Invest 68:492–500
Nowell PC, Hungerford DA (1960) A minute chromosome in human chronic granulocytic leukemia. Science 132:1497–1499
Rowley JD (1973) A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature 243:290–293
Shtivelman E, Lifshitz B, Gale RP, Roe BA, Canaani E (1986) Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene. Cell 47:277–284
Calabretta B, Perrotti D (2004) The biology of CML blast crisis. Blood 103:4010–4022
Druker BJ, Guilhot F, O’Brien SG, Gathmann I, Kantarjian H, Gattermann N, Deininger MW, Silver RT, Goldman JM, Stone RM, Cervantes F, Hochhaus A, Powell BL, Gabrilove JL, Rousselot P, Reiffers J, Cornelissen JJ, Hughes T, Agis H, Fischer T, Verhoef G, Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich JP, Simonsson B, Taylor K, Baccarani M, So C, Letvak L, Larson RA (2006) Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia. N Engl J Med 355:2408–2417
Gorre ME, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao PN, Sawyers CL (2001) Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293:876–880
O’Hare T, Eide CA, Deininger MW (2007) Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood 110:2242–2249
Lombardo LJ, Lee FY, Chen P, Norris D, Barrish JC, Behnia K, Castaneda S, Cornelius LA, Das J, Doweyko AM, Fairchild C, Hunt JT, Inigo I, Johnston K, Kamath A, Kan D, Klei H, Marathe P, Pang S, Peterson R, Pitt S, Schieven GL, Schmidt RJ, Tokarski J, Wen ML, Wityak J, Borzilleri RM (2004) Discovery of N-(2-chloro-6-methyl- phenyl)-2-(6-(4-(2-hydroxyethyl)- piperazin-1-yl)-2-methylpyrimidin-4- ylamino)thiazole-5-carboxamide (BMS-354825), a dual Src/Abl kinase inhibitor with potent antitumor activity in preclinical assays. J Med Chem 47:6658–6661
Talpaz M, Shah NP, Kantarjian H, Donato N, Nicoll J, Paquette R, Cortes J, O’Brien S, Nicaise C, Bleickardt E, Blackwood-Chirchir MA, Iyer V, Chen TT, Huang F, Decillis AP, Sawyers CL (2006) Dasatinib in imatinib-resistant Philadelphia chromosome-positive leukemias. N Engl J Med 354:2531–2541
Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF (1987) Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia. Nature 328:342–344
Thery C, Zitvogel L, Amigorena S (2002) Exosomes: composition, biogenesis and function. Nat Rev Immunol 2:569–579
Cocucci E, Racchetti G, Meldolesi J (2009) Shedding microvesicles: artefacts no more. Trends Cell Biol 19:43–51
Thery C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593
Lakkaraju A, Rodriguez-Boulan E (2008) Itinerant exosomes: emerging roles in cell and tissue polarity. Trends Cell Biol 18:199–209
Webber J, Steadman R, Mason MD, Tabi Z, Clayton A (2010) Cancer exosomes trigger fibroblast to myofibroblast differentiation. Cancer Res 70:9621–9630
Szajnik M, Czystowska M, Szczepanski MJ, Mandapathil M, Whiteside TL (2010) Tumor-derived microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg). PLoS One 5:e11469
Skog J, Wurdinger T, van Rijn S, Meijer DH, Gainche L, Sena-Esteves M, Curry WT Jr, Carter BS, Krichevsky AM, Breakefield XO (2008) Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol 10:1470–1476
Gesierich S, Berezovskiy I, Ryschich E, Zoller M (2006) Systemic induction of the angiogenesis switch by the tetraspanin D6.1A/CO-029. Cancer Res 66:7083–7094
Savina A, Furlan M, Vidal M, Colombo MI (2003) Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J Biol Chem 278:20083–20090
Doong H, Rizzo K, Fang S, Kulpa V, Weissman AM, Kohn EC (2003) CAIR-1/BAG-3 abrogates heat shock protein-70 chaperone complex-mediated protein degradation: accumulation of poly-ubiquitinated Hsp90 client proteins. J Biol Chem 278:28490–28500
Onfelt B, Nedvetzki S, Benninger RK, Purbhoo MA, Sowinski S, Hume AN, Seabra MC, Neil MA, French PM, Davis DM (2006) Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria. J Immunol 177:8476–8483
Thomas SM, Brugge JS (1997) Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13:513–609
Folkman J, Shing Y (1992) Angiogenesis. J Biol Chem 267:10931–10934
Taverna S, Flugy A, Saieva L, Kohn EC, Santoro A, Meraviglia S, De Leo G, Alessandro R (2011) Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis. Int J Cancer. doi:10.1002/ijc.26217
Gerdes HH, Carvalho RN (2008) Intercellular transfer mediated by tunneling nanotubes. Curr Opin Cell Biol 20:470–475
Rustom A, Saffrich R, Markovic I, Walther P, Gerdes HH (2004) Nanotubular highways for intercellular organelle transport. Science 303:1007–1010
Sowinski S, Jolly C, Berninghausen O, Purbhoo MA, Chauveau A, Kohler K, Oddos S, Eissmann P, Brodsky FM, Hopkins C, Onfelt B, Sattentau Q, Davis DM (2008) Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission. Nat Cell Biol 10:211–219
Koyanagi M, Brandes RP, Haendeler J, Zeiher AM, Dimmeler S (2005) Cell-to-cell connection of endothelial progenitor cells with cardiac myocytes by nanotubes: a novel mechanism for cell fate changes? Circ Res 96:1039–1041
Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J, Lydon NB (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 2:561–566
Legros L, Bourcier C, Jacquel A, Mahon FX, Cassuto JP, Auberger P, Pages G (2004) Imatinib mesylate (STI571) decreases the vascular endothelial growth factor plasma concentration in patients with chronic myeloid leukemia. Blood 104:495–501
Cortes J, Hochhaus A, Hughes T, Kantarjian H (2011) Front-line and salvage therapies with tyrosine kinase inhibitors and other treatments in chronic myeloid leukemia. J Clin Oncol 29:524–531
Kanda S, Miyata Y, Kanetake H, Smithgall TE (2007) Non-receptor protein-tyrosine kinases as molecular targets for antiangiogenic therapy (Review). Int J Mol Med 20:113–121
Mukhopadhyay D, Tsiokas L, Sukhatme VP (1995) Wild-type p53 and v-Src exert opposing influences on human vascular endothelial growth factor gene expression. Cancer Res 55:6161–6165
Kim LC, Song L, Haura EB (2009) Src kinases as therapeutic targets for cancer. Nat Rev Clin Oncol 6:587–595
Summy JM, Trevino JG, Lesslie DP, Baker CH, Shakespeare WC, Wang Y, Sundaramoorthi R, Metcalf CA 3rd, Keats JA, Sawyer TK, Gallick GE (2005) AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes. Mol Cancer Ther 4:1900–1911
Masiero L, Lapidos KA, Ambudkar I, Kohn EC (1999) Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx. J Cell Sci 112(Pt 19):3205–3213
Acknowledgments
This work was supported by the Intramural Program of the Center for Cancer Research, NCI; Dr. Mineo was supported by a fellowship from Italian Association for Cancer Research (AIRC). The authors thank Drs. Virador and Muller for assistance with electron microscopy, and Mr. Lim and Ms. Mannan for confocal microscopy technical assistance.
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10456_2011_9241_MOESM1_ESM.tif
K562 exosomes are preferentially taken up by endothelial cells. HUVECs (a, b) and CCD27sK fibroblasts (c, d) were plated on glass and incubated for 4 h with PKH26-labeled exosomes (red). After the incubation, cell were fixed and stained for nuclei (blue) and actin (green). Scale bars: 20 μm (TIFF 1096 kb)
Transfer of PKH26-stained K562 exosomes through nanotubes connecting HUVECs. Exosomes were labeled with PKH26, incubated with HUVECs on Matrigel, and after 3 h, the cells were monitored by time-lapse video-microscopy. Movie 1 was acquired at 1 frame/15 s for a period of 5.5 min. Movie 2 was acquired at 1 frame/30 s for a period of 25 min (MPG 48634 kb)
Transfer of PKH26-stained K562 exosomes through nanotubes connecting HUVECs. Exosomes were labeled with PKH26, incubated with HUVECs on Matrigel, and after 3 h, the cells were monitored by time-lapse video-microscopy. Movie 1 was acquired at 1 frame/15 s for a period of 5.5 min. Movie 2 was acquired at 1 frame/30 s for a period of 25 min (MPG 99193 kb)
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Mineo, M., Garfield, S.H., Taverna, S. et al. Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a src-dependent fashion. Angiogenesis 15, 33–45 (2012). https://doi.org/10.1007/s10456-011-9241-1
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DOI: https://doi.org/10.1007/s10456-011-9241-1