Abstract
The establishment of a vascular network within tumours is a key step in the progression towards an aggressive, metastatic state, with poor prognosis. We have developed a novel in vitro model to specifically capture the interaction between endothelial cells and solid tumours. Micro-vascularised in vitro tumour constructs were produced by introducing endothelial cells to multicellular spheroids formed in hanging drops. Upon introduction, the endothelial cells migrated into the tumour spheroid, establishing tubular networks and luminal structures. This system relies on the natural pro-angiogenic capacity of multicellular spheroids, and does not require the addition of exogenous angiogenic factors, or use of extracellular-matrix substitutes.
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Beecken W, Kramer W, Jonas D et al. New molecular mediators in tumor angiogenesis. J Cell Mol Med 2000; 4(4): 262–269.
Gourley M, Williamson J. Angiogenesis: New targets for the development of anticancer chemotherapies. Curr Pharm Des 2000; 6(4): 417–39.
Kerbel R. Tumor angiogenesis: Past, present and the near future. Carcinogenesis 2000; 21(3): 505–15.
Fidler I. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat Rev Cancer 2003; 3(6): 453–8.
Fontanini G, Lucchi M, Vignati S et al. Angiogenesis as a prognostic indicator of survival in non-small-cell lung carcinoma: A prospective study. J Natl Cancer Inst 1997; 89(12): 881–6.
Koomagi R, Volm M. Tissue-factor expression in human non-smallcell lung carcinoma measured by immunohistochemistry: correlation between tissue factor and angiogenesis. Int J Cancer 1998; 79(1): 19–22.
Hamada K, Kuratsu J, Saitoh Y. Expression of tissue factor correlates with grade of malignancy in human glioma. Cancer 1996; 77(9): 1877–83.
Nakasaki T, Wada H, Shigemori C et al. Expression of tissue factor and vascular endothelial growth factor is associated with angiogenesis in colorectal cancer. Am J Hematol 2002; 69(4): 247–54.
Kaku T, Kamura T, Kinukawa N et al. Angiogenesis in endometrial carcinoma. Cancer 1997; 80(4): 741–7.
Vermeulen P, Gasparini G, Fox S. Quantification of angiogenesis in solid human tumours: An international consensus on the methodology and criteria of evaluation. Eur J Cancer 1996; 32A(14): 2474–84.
Genentech: 2004, ‘Avastin (bevacizumab)'. http://www.gene.com/gene/ pipeline/status/oncology/avastin/index.jsp.
Sato Y. Molecular diagnosis of tumour angiogenesis and anti-angiogenic cancer therapy. Int J Clin Oncol 2003; 8: 200–6.
McCarthy M. Antiangiogenesis drug promising for metastatic colorectal cancer. Lancet 2003; 361(9373): 1959.
Auerbach R, Akhtar N, Lewis R, Shinners B. Angiogenesis assays: problems and pitfalls. Cancer Metastasis Rev 2003; 19 (1–2) 167–72.
Vernon R, Gooden M. New technologies in vitro for analysis of cell movement on or within collagen gels. Matrix Biol 2002; 21(8): 661–9.
Vernon R, Sage E. A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices. Microvasc Res 1999; 57(2): 118–33.
Donovan D, Brown NJ, Bishop ET, Lewis CE. Comparison of three in vitro human ‘angiogenesis’ assays with capillaries formed in vivo. Angiogenesis: 2001; 4(2): 113–21.
Montaez E, Casaroli-Marano R, Vilar S, Pagan R. Comparative study of tube assembly, in three-dimensional collagne matix and on Matrigel coats. Angiogenesis 2002; 5: 167–72.
Auerbach R, Lewis R, Shinners B et al. Angiogenesis assays: A critical overview. Clin Chem 2003; 49 (1): 32–40.
Vailhe B, Vittet D, Feige J et al. In vitro models of vasculogenesis and angiogenesis. Lab Invest 2001; 81(4): 439–52.
Bishop E, Bell G, Bloor S et al. An in vitro model of angiogenesis: Basic features. Angiogenesis 1999; 3: 335–44.
Ment L, Stewart W, Scaramuzzino D, Madri J. An in vitro threedimensional coculture model of cerebral microvascular angiogenesis and differentiation. In Vitro Cell Dev Biol Anim 1997; 33(9): 684–91.
Korff T, Kimmina S, Martiny-Baron G, Augustin HG. Blood vessel maturation in a 3-dimensional spheroidal coculture model: Direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness. FASEB J 2001; 15(2): 447–57.
Korff T, Augustin H. Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J Cell Biol 1998; 143(5): 1341–52.
Haspel H, Scicli G, McMahon G, Scicli A. Inhibition of vascular endothelial growth factor-associated tyrosine kinase activity with SU5416 blocks sprouting in the microvascular endothelial cell spheroid model of angiogenesis. Microvasc Res 2002; 63(3): 304–15.
Kozien D, Gerol M, Hendey B, RayChaudhury A. A novel in vitro model of tumor angiogenesis. In Vitro Cell Dev Biol Anim 2000; 36(9): 555–8.
Oudar O. Spheroids: Relation between tumour and endothelial cells. Crit Rev Oncol Hematol 2000; 36(2–3): 99–106.
Chopra V, Dinh TV, Hannigan EV. Three-dimensional endothelialtumor epithelial cell interactions in human cervical cancers. In Vitro Cell Dev Biol Anim 1997; 33(6): 432–42.
Knuchel R, Feichtinger J, Recktenwald A et al. Interactions between bladder tumor cells as tumor spheroids from the cell line J82 and human endothelial cells in vitro. J Urol 1988; 139(3): 640–5.
Offner F, Bigalke I, Schiefer J et al. Interaction of human malignant melanoma tumor spheroids with endothelium and reconstituted basement membrane: Modulation by RGDS. Int J Cancer 1993; 54(3): 506–12.
Kelm J, Timmins N, Brown C et al. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 2003; 83(2): 173–80.
Wartenberg M, Dnmez F, Ling F et al. Tumour-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells. FASEB J 2001; 15: 995–1005.
Shweiki D, Neeman M, Itin A, Keshet E. Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: Implications for tumor angiogenesis. Proc Natl Acad Sci USA 1995; 92(3): 768–72.
Lazareff J, Suwinski R, De Rosa R, Olmstead C. Tumor volume and growth kinetics in hypothalamic-chiasmatic pediatric low grade gliomas. Pediatr Neurosurg 1999; 30(6): 312–9.
Marusic M, Bajzer Z, Vuk-Pavlovic, Freyer J. Tumour growth in vivo and as multicellualr spheroids compared by mathematical models. Bull Math Biol 1994; 56: 617–631.
Kunz-Schughart L, Mueller-Klieser W. Three-dimensional culture. In. Masters J (ed): Animal Cell Culture: A Practical Approach. New York: Oxford University Press, 3rd edition, 2000; 123–84.
Hemmerlein B, Kugler A, Ozisik R et al. Vascular endothelial growth factor expression, angiogenesis, and necrosis in renal cell carcinomas. Virchows Arch 2001; 439(5): 645–52.
Guan M, Jin J, Su B et al. Tissue factor expression and angiogenesis in human glioma. Clin Biochem 2002; 35(4): 321–5.
Sonoda T, Kobayashi H, Kaku T et al. Expression of angiogenesis factors in monolayer culture, multicellular spheroid and in vivo transplanted tumor by human ovarian cancer cell lines. Cancer Lett 2003; 196(2): 229–37.
Burns P, Wilson D. Angiogenesis mediated by metabolites is dependent on vascular endothelial growth factor (VEGF). Angiogenesis 2003; 6(1): 73–7.
Pavlakovic H, Havers W, Schweigerer L. Multiple angiogenesis stimulators in a single malignancy: Implications for anti-angiogenic tumour therapy. Angiogenesis 2001; 4(4): 259–62.
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Timmins, N., Dietmair, S. & Nielsen, L. Hanging-drop multicellular spheroids as a model of tumour angiogenesis. Angiogenesis 7, 97–103 (2004). https://doi.org/10.1007/s10456-004-8911-7
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DOI: https://doi.org/10.1007/s10456-004-8911-7