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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 27, 2004 as doi:10.1096/fj.04-1934fje. |
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Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
1Correspondence: 16-560, Biological Engineering Division, M.I.T., 77 Massachusetts Ave, Cambridge, MA 02139, USA. E-mail: rams{at}MIT.edu
SPECIFIC AIMS
Our purpose was to develop a 3-dimensional dual fluorescence imaging-based in vitro culture system that allows the dissection of angiotherapeutics from chemotherapeutics, integrating the extracellular matrix as a key component modulating the outcome.
PRINCIPAL FINDINGS
1. VEGF and HGF induce tumor angiogenesis in vitro, which can be pharmacologically dissected at the receptor level using PTK787
Endothelial cells formed a limited number of tubular networks within 24 h of plating on matrigel (1:3 dilution). However, the addition of tumor cells to establish the coculture accelerated the tubulogenic process. The GFP+ tumor cells were visualized to concentrate into clusters surrounded and integrating with the vascular network. Addition of both VEGF and HGF/SF resulted in a significant increase in the vascular network. To validate the sensitivity of the system to elucidate the modulation of specific pathways, we used a VEGF receptor antagonist, PTK787. As expected, VEGF-induced angiogenesis was blocked by PTK787 at a concentration that had no effect on the HGF/SF-induced response.
2. Doxorubicin selectively ablates the melanoma cells, while the antiangiogenic outcome of combretastatin and thalidomide depends on the angiogenic growth factors
Incubation of the coculture, plated on matrigel, with doxorubicin (10–50 µM) exerted a selective ablation of the tumor cells in a concentration-dependent manner. Even at the highest concentration used (50 µM), no effect on the VEGF-induced endothelial network was evident. In contrast, both thalidomide and combretastatin exerted a collapse of the VEGF-induced vascular network without affecting the tumor cells.
Similar to the VEGF-induced coculture experiments, doxorubicin exerted a selective induction of tumor cell death in the presence of HGF/SF (Fig. 1
). In contrast to VEGF, HGF/SF prevented the ablation of endothelial cellular network in the presence of thalidomide or combretastatin (Fig. 1)
.
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3. The extracellular matrix impinges on the antiangiogenic outcome of combretastatin and thalidomide in VEGF-or HGF-induced tumor response
Endothelial cells plated on collagen matrix assumed a flat "cobblestone" morphology unlike the tubular networks formed when plated on matrigel. The melanoma cells also assumed a "spreading-out" morphology with the formations of focal adhesions, and did not form cell clusters as seen on matrigel. Incubation with doxorubicin induced tumor cell death in both VEGF- and HGF/SF-treated cocultures (Fig. 2
). As shown in Fig. 2
, both combretastatin and thalidomide inhibited the angiogenic effects of VEGF. The protective effect of HGF/SF observed on cells plated on matrigel was lost when the cells were plated on collagen, and both thalidomide and combretastatin induced endothelial cell loss (Fig. 2)
.
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CONCLUSIONS
Angiogenesis, the development of new blood vessels from an existing vascular bed, underlies the rapid expansion of tumors and the development of distant metastasis. Executed in distinct sequential steps, angiogenesis is the culmination of spatio-temporal interactions between the tumor cells, the extracellular matrix, and the endothelial cells brought about by the interplay of multiple mediators. The understanding of the events underlying this complex process and the elucidation of the mechanisms of action of some of the mediators has opened up the possibility of therapeutic targeting of angiogenesis as a novel strategy for tumor management, with over sixty compounds in clinical stages of development. However, recent experiences have pointed toward more of an adjunct role for this approach in cohesion with standard chemotherapy, emphasizing the need for information from models that allow the integration of the parenchyma-stroma axis.
Here we report the development of a 3-dimensional dual fluorescence imaging-based in vitro culture system that allows the dissection of angiotherapeutics from chemotherapeutics, integrating the extracellular matrix as a key component modulating the outcome. In a recent study, we demonstrated that hepatocyte growth factor/scatter factor (HGF/SF) exerts a potent angiogenic effect even in the presence of a vascular endothelial growth factor (VEGF) receptor blockade. The observation in this study was the distinction in the angiogenic activity of VEGF and HGF/SF, which are tyrosine kinase receptor ligands signaling through the VEGF receptor family and the MET receptor, respectively. The distinction at the receptor level was demonstrated by the selective inhibition of VEGF activity in the presence of PTK787, a VEGF receptor antagonist, which is in advanced stages of development for its antiangiogenic indications. The other distinction was in the susceptibility of VEGF- and HGF/SF-induced angiogenesis to modulation by known antiangiogenics, combretastatin and thalidomide. The susceptibility of VEGF-induced angiogenesis and the protective effect of HGF/SF against these two indirect antiangiogenics indicate the functional difference at the level of intracellular signaling induced by the two growth factors. The loss of function of HGF/SF when the ECM is altered suggests that the extracellular environment impinges on the outcome (Fig. 3
). The current model encompasses the possibility of studying tumor-angiogenesis-matrix interaction on the same platform, expanding the high throughput screening of effective regimens combining chemotherapeutics and antiangiogenics in a simulated clinicopathological setting.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1934fje;
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