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Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration Across Brain and Lung Microvascular Endothelium

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Abstract

Tumor cell extravasation through the endothelial barrier forming the microvessel wall is a crucial step during tumor metastasis. However, where, how and how fast tumor cells transmigrate through endothelial barriers remain unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through brain and lung microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in the structural components (integrity) of endothelial monolayers were quantified by confocal microscopy. Endothelial monolayer permeability to albumin P albumin was also quantified under the same conditions. We found that about 98% of transmigration occurred at the joints of endothelial cells instead of cell bodies; tumor cell adhesion and transmigration degraded endothelial surface glycocalyx and disrupted endothelial junction proteins, consequently increased P albumin; more tumor cells adhered to and transmigrated through the endothelial monolayer with higher P albumin; P albumin and tumor transmigration were increased by vascular endothelial growth factor, a representative of cytokines, and lipopolysaccharides, a typical systemic inflammatory factor, but reduced by adenosine 3′,5′-cyclic monophosphate. These results suggest that reinforcing endothelial structural integrity is an effective approach for inhibiting tumor extravasation.

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References

  1. Adamson, R. H., J. C. Ly, R. K. Sarai, J. F. Lenz, A. Altangerel, D. Drenckhahn, and F. E. Curry. Epac/Rap1 pathway regulates microvascular hyperpermeability induced by PAF in rat mesentery. Am. J. Physiol. Heart Circ. Physiol. 294:H1188–H1196, 2008.

    Article  CAS  PubMed  Google Scholar 

  2. Al-Mehdi, A. B., K. Tozawa, A. B. Fisher, L. Shientag, A. Lee, and R. J. Muschel. Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat. Med. 6:100–102, 2000.

    Article  CAS  PubMed  Google Scholar 

  3. Arvanitis, C., S. Khuon, R. Spann, K. M. Ridge, and T. L. Chew. Structure and biomechanics of the endothelial transcellular circumferential invasion array in tumor invasion. PLoS One 9:e89758, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bates, D. O. Vascular endothelial growth factors and vascular permeability. Cardiovasc. Res. 87:262–271, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bendas, G., and L. Borsig. Cancer cell adhesion and metastasis: selectins, integrins, and the inhibitory potential of heparins. Int. J. Cell Biol. 2012:676731, 2012.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Brenner, W., P. Langer, F. Oesch, C. J. Edgell, and R. J. Wieser. Tumor cell–endothelium adhesion in an artificial venule. Anal. Biochem. 225:213–219, 1995.

    Article  CAS  PubMed  Google Scholar 

  7. Burns, A. R., D. C. Walker, E. S. Brown, L. T. Thurmon, R. A. Bowden, C. R. Keese, S. I. Simon, M. L. Entman, and C. W. Smith. Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners. J Immunol. 159:2893–2903, 1997.

    CAS  PubMed  Google Scholar 

  8. Cai, B., J. Fan, M. Zeng, L. Zhang, and B. M. Fu. Adhesion of malignant mammary tumor cells MDA-MB-231 to microvessel wall increases microvascular permeability via degradation of endothelial surface glycocalyx. J. Appl. Physiol. 113:1141–1153, 2012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chen, C., and D. B. Khismatullin. Lipopolysaccharide induces the interactions of breast cancer and endothelial cells via activated monocytes. Cancer Lett. 345:75–84, 2014.

    Article  CAS  PubMed  Google Scholar 

  10. Chen, M. B., J. A. Whisler, J. S. Jeon, and R. D. Kamm. Mechanisms of tumor cell extravasation in an in vitro microvascular network platform. Integr. Biol. (Camb). 5:1262–1271, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cheung, L. S., P. S. Raman, E. M. Balzer, D. Wirtz, and K. Konstantopoulos. Biophysics of selectin-ligand interactions in inflammation and cancer. Phys. Biol. 8:015013, 2011.

    Article  PubMed  Google Scholar 

  12. Chotard-Ghodsnia, R., O. Haddad, A. Leyrat, A. Drochon, C. Verdier, and A. Duperray. Morphological analysis of tumor cell/endothelial cell interactions under shear flow. J. Biomech. 40:335–344, 2007.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Cinamon, G., and R. Alon. A real time in vitro assay for studying leukocyte transendothelial migration under physiological flow conditions. J. Immunol. Methods 273:53–62, 2003.

    Article  CAS  PubMed  Google Scholar 

  14. Constantinescu, A. A., H. Vink, and J. A. Spaan. Endothelial cell glycocalyx modulates immobilization of leukocytes at the endothelial surface. Arterioscler. Thromb. Vasc. Biol. 23:1541–1547, 2003.

    Article  CAS  PubMed  Google Scholar 

  15. Curry, F. R., and R. H. Adamson. Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge. Cardiovasc. Res. 87:218–229, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Curry, F. R., and T. Noll. Spotlight on microvascular permeability. Cardiovasc. Res. 87:195–197, 2010.

    Article  CAS  PubMed  Google Scholar 

  17. Earley, S., and G. E. Plopper. Disruption of focal adhesion kinase slows transendothelial migration of AU-565 breast cancer cells. Biochem. Biophys. Res. Commun. 350:405–412, 2006.

    Article  CAS  PubMed  Google Scholar 

  18. Eichler, A. F., E. Chung, D. P. Kodack, J. S. Loeffler, D. Fukumura, and R. K. Jain. The biology of brain metastases-translation to new therapies. Nat. Rev. Clin. Oncol. 8:344–356, 2011.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Fan, J., B. Cai, M. Zeng, Y. Hao, F. G. Giancotti, and B. M. Fu. Integrin beta4 signaling promotes mammary tumor cell adhesion to brain microvascular endothelium by inducing ErbB2-mediated secretion of VEGF. Ann. Biomed. Eng. 39:2223–2241, 2011.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Fazakas, C., I. Wilhelm, P. Nagyoszi, A. E. Farkas, J. Hasko, J. Molnar, H. Bauer, H. C. Bauer, F. Ayaydin, N. T. Dung, L. Siklos, and I. A. Krizbai. Transmigration of melanoma cells through the blood-brain barrier: role of endothelial tight junctions and melanoma-released serine proteases. PLoS One 6:e20758, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Fertig, E. J., E. Lee, N. B. Pandey, and A. S. Popel. Analysis of gene expression of secreted factors associated with breast cancer metastases in breast cancer subtypes. Sci. Rep. 5:12133, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fidler, I. J. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat. Rev. Cancer 3:453–458, 2003.

    Article  CAS  PubMed  Google Scholar 

  23. Fu, B. M., S. Shen, and B. Chen. Structural mechanisms in the abolishment of VEGF-induced microvascular hyperpermeability by cAMP. J. Biomech. Eng. 128:317–328, 2006.

    PubMed  Google Scholar 

  24. Fu, B. M., and J. M. Tarbell. Mechano-sensing and transduction by endothelial surface glycocalyx: composition, structure, and function. Wiley Interdiscip. Rev. Syst. Biol. Med. 5:381–390, 2013.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ghaly, T., M. M. Rabadi, M. Weber, S. M. Rabadi, M. Bank, J. M. Grom, J. T. Fallon, M. S. Goligorsky, and B. B. Ratliff. Hydrogel-embedded endothelial progenitor cells evade LPS and mitigate endotoxemia. Am. J. Physiol. Renal. Physiol. 301:F802–F812, 2011.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Glinskii, O. V., V. H. Huxley, G. V. Glinsky, K. J. Pienta, A. Raz, and V. V. Glinsky. Mechanical entrapment is insufficient and intercellular adhesion is essential for metastatic cell arrest in distant organs. Neoplasia. 7:522–527, 2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Glinskii, O. V., F. Li, L. S. Wilson, S. Barnes, K. Rittenhouse-Olson, J. J. Barchi, Jr, K. J. Pienta, and V. V. Glinsky. Endothelial integrin alpha3beta1 stabilizes carbohydrate-mediated tumor/endothelial cell adhesion and induces macromolecular signaling complex formation at the endothelial cell membrane. Oncotarget. 5:1382–1389, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Haddad, O., R. Chotard-Ghodsnia, C. Verdier, and A. Duperray. Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFkappaB: differential role of the shear stress. Exp. Cell Res. 316:615–626, 2010.

    Article  CAS  PubMed  Google Scholar 

  29. Hamilla, S. M., K. M. Stroka, and H. Aranda-Espinoza. VE-cadherin-independent cancer cell incorporation into the vascular endothelium precedes transmigration. PLoS One 9:e109748, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Heyder, C., E. Gloria-Maercker, F. Entschladen, W. Hatzmann, B. Niggemann, K. S. Zanker, and T. Dittmar. Realtime visualization of tumor cell/endothelial cell interactions during transmigration across the endothelial barrier. J. Cancer Res. Clin. Oncol. 128:533–538, 2002.

    Article  CAS  PubMed  Google Scholar 

  31. Heyder, C., E. Gloria-Maercker, W. Hatzmann, K. S. Zaenker, and T. Dittmar. Visualization of tumor cell extravasation. Contrib. Microbiol. 13:200–208, 2006.

    Article  PubMed  Google Scholar 

  32. Jeon, J. S., S. Bersini, M. Gilardi, G. Dubini, J. L. Charest, M. Moretti, and R. D. Kamm. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc. Natl. Acad. Sci. USA 112:214–219, 2015.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kienast, Y., L. von Baumgarten, M. Fuhrmann, W. E. Klinkert, R. Goldbrunner, J. Herms, and F. Winkler. Real-time imaging reveals the single steps of brain metastasis formation. Nat. Med. 16:116–122, 2010.

    Article  CAS  PubMed  Google Scholar 

  34. Kuperwasser, C., S. Dessain, B. E. Bierbaum, D. Garnet, K. Sperandio, G. P. Gauvin, S. P. Naber, R. A. Weinberg, and M. Rosenblatt. A mouse model of human breast cancer metastasis to human bone. Cancer Res. 65:6130–6138, 2005.

    Article  CAS  PubMed  Google Scholar 

  35. Lee, T. H., H. K. Avraham, S. Jiang, and S. Avraham. Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability. J. Biol. Chem. 278:5277–5284, 2003.

    Article  CAS  PubMed  Google Scholar 

  36. Li, G., M. J. Simon, L. M. Cancel, Z. D. Shi, X. Ji, J. M. Tarbell, B. Morrison, 3rd, and B. M. Fu. Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies. Ann. Biomed. Eng. 38:2499–2511, 2010.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Miles, F. L., F. L. Pruitt, K. L. van Golen, and C. R. Cooper. Stepping out of the flow: capillary extravasation in cancer metastasis. Clin. Exp. Metastasis 25:305–324, 2008.

    Article  CAS  PubMed  Google Scholar 

  38. Mulivor, A. W., and H. H. Lipowsky. Inhibition of glycan shedding and leukocyte-endothelial adhesion in postcapillary venules by suppression of matrixmetalloprotease activity with doxycycline. Microcirculation. 16:657–666, 2009.

    Article  CAS  PubMed  Google Scholar 

  39. Muller, A., B. Homey, H. Soto, N. Ge, D. Catron, M. E. Buchanan, T. McClanahan, E. Murphy, W. Yuan, S. N. Wagner, J. L. Barrera, A. Mohar, E. Verastegui, and A. Zlotnik. Involvement of chemokine receptors in breast cancer metastasis. Nature 410:50–56, 2001.

    Article  CAS  PubMed  Google Scholar 

  40. Nguyen, D. X., P. D. Bos, and J. Massague. Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer 9:274–284, 2009.

    Article  CAS  PubMed  Google Scholar 

  41. Peyssonnaux, C., P. Cejudo-Martin, A. Doedens, A. S. Zinkernagel, R. S. Johnson, and V. Nizet. Cutting edge: essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis. J Immunol. 178:7516–7519, 2007.

    Article  CAS  PubMed  Google Scholar 

  42. Pries, A. R., T. W. Secomb, and P. Gaehtgens. The endothelial surface layer. Pflugers Arch. 440:653–666, 2000.

    Article  CAS  PubMed  Google Scholar 

  43. Reitsma, S., D. W. Slaaf, H. Vink, M. A. van Zandvoort, and M. G. Oude Egbrink. The endothelial glycocalyx: composition, functions, and visualization. Pflugers Arch. 454:345–359, 2007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Schick, M. A., C. Wunder, J. Wollborn, N. Roewer, J. Waschke, C. T. Germer, and N. Schlegel. Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation. J. Physiol. 590:2693–2708, 2012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Schlegel, N., and J. Waschke. cAMP with other signaling cues converges on Rac1 to stabilize the endothelial barrier—a signaling pathway compromised in inflammation. Cell Tissue Res. 355:587–596, 2014.

    Article  CAS  PubMed  Google Scholar 

  46. Schmidt, E. P., Y. Yang, W. J. Janssen, A. Gandjeva, M. J. Perez, L. Barthel, R. L. Zemans, J. C. Bowman, D. E. Koyanagi, Z. X. Yunt, L. P. Smith, S. S. Cheng, K. H. Overdier, K. R. Thompson, M. W. Geraci, I. S. Douglas, D. B. Pearse, and R. M. Tuder. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nat. Med. 18:1217–1223, 2012.

    Article  CAS  PubMed  Google Scholar 

  47. Schumacher, D., B. Strilic, K. K. Sivaraj, N. Wettschureck, and S. Offermanns. Platelet-derived nucleotides promote tumor-cell transendothelial migration and metastasis via P2Y2 receptor. Cancer Cell 24:130–137, 2013.

    Article  CAS  PubMed  Google Scholar 

  48. Shen, S., J. Fan, B. Cai, Y. Lv, M. Zeng, Y. Hao, F. G. Giancotti, and B. M. Fu. Vascular endothelial growth factor enhances cancer cell adhesion to microvascular endothelium in vivo. Exp. Physiol. 95:369–379, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Shi, L., M. Zeng, Y. Sun, and B. M. Fu. Quantification of blood-brain barrier solute permeability and brain transport by multiphoton microscopy. J. Biomech. Eng. 136:031005, 2014.

    Article  PubMed  Google Scholar 

  50. Slattery, M. J., S. Liang, and C. Dong. Distinct role of hydrodynamic shear in leukocyte-facilitated tumor cell extravasation. Am. J. Physiol. Cell Physiol. 288:C831–C839, 2005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Steeg, P. S. Tumor metastasis: mechanistic insights and clinical challenges. Nat. Med. 12:895–904, 2006.

    Article  CAS  PubMed  Google Scholar 

  52. Stoletov, K., H. Kato, E. Zardouzian, J. Kelber, J. Yang, S. Shattil, and R. Klemke. Visualizing extravasation dynamics of metastatic tumor cells. J. Cell Sci. 123:2332–2341, 2010.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Tarbell, J. M., and M. Y. Pahakis. Mechanotransduction and the glycocalyx. J. Intern. Med. 259:339–350, 2006.

    Article  CAS  PubMed  Google Scholar 

  54. Thorley, A. J., P. A. Ford, M. A. Giembycz, P. Goldstraw, A. Young, and T. D. Tetley. Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages. J Immunol. 178:463–473, 2007.

    Article  CAS  PubMed  Google Scholar 

  55. Tremblay, P. L., F. A. Auger, and J. Huot. Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases. Oncogene 25:6563–6573, 2006.

    Article  CAS  PubMed  Google Scholar 

  56. Tremblay, P. L., J. Huot, and F. A. Auger. Mechanisms by which E-selectin regulates diapedesis of colon cancer cells under flow conditions. Cancer Res. 68:5167–5176, 2008.

    Article  CAS  PubMed  Google Scholar 

  57. Vlodavsky, I., M. Elkin, G. Abboud-Jarrous, F. Levi-Adam, L. Fuks, I. Shafat, and N. Ilan. Heparanase: one molecule with multiple functions in cancer progression. Connect. Tissue Res. 49:207–210, 2008.

    Article  CAS  PubMed  Google Scholar 

  58. Weidert, E., S. E. Pohler, E. W. Gomez, and C. Dong. Actinomyosin contraction, phosphorylation of VE-cadherin, and actin remodeling enable melanoma-induced endothelial cell-cell junction disassembly. PLoS One 9:e108092, 2014.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Weis, S., J. Cui, L. Barnes, and D. Cheresh. Endothelial barrier disruption by VEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. J. Cell Biol. 167:223–229, 2004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Zen, K., D. Q. Liu, Y. L. Guo, C. Wang, J. Shan, M. Fang, C. Y. Zhang, and Y. Liu. CD44v4 is a major E-selectin ligand that mediates breast cancer cell transendothelial migration. PLoS One 3:e1826, 2008.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the NIH/NCI Grant SC1CA153325-01.

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  1. Department of Biomedical Engineering, The City College of the City University of New York, 160 Convent Ave, New York, NY, 10031, USA

    Jie Fan & Bingmei M. Fu

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Fan, J., Fu, B.M. Quantification of Malignant Breast Cancer Cell MDA-MB-231 Transmigration Across Brain and Lung Microvascular Endothelium. Ann Biomed Eng 44, 2189–2201 (2016). https://doi.org/10.1007/s10439-015-1517-y

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