ABSTRACT
The most well-known approach developed to target tumor angiogenesis includes drugs that neutralize pro-angiogenic growth factors such as VEGF, or block signaling through VEGFRs. Currently, all approved anti-angiogenic drugs either target VEGF or VEGFR tyrosine kinase receptors. The concept of ‘accidental’ angiogenic inhibitors for the treatment of cancer refers to the idea that many anticancer drugs, both old and new, not developed with the intention of inhibiting angiogenesis, may in fact do so, thus contributing to their overall anti-tumor effects (Kerbel et al. 2000). An anti-angiogenic effect was even observed with traditional chemotherapeutic agents (Miller et al. 2001). Many in vitro and in vivo methods currently exist to assess angiogenesis. In vitro assays for measuring angiogenesis mainly use isolated endothelial cells and allow reproducible measurement of a portion of the angiogenic process. There are also assays which incorporate multiple cell types, such as the aortic ring assay (Nicosia et al. 1982). However, these assays focus only on early steps in angiogenesis do not take into account the remodeling and stabilization which occur once flow begins in a vessel. While cell lines are well utilized in angiogenesis research, results from cell lines rarely correlate with the anti-angiogenic potential of these drugs in vivo. There are a number of other in vivo assays to measure angiogenesis. The oldest of these use transparent chambers to allow growing blood vessels to be visualized microscopically (Sandison 1924). More recent assays measuring vessel ingrowth into subcutaneous implants such as matrigel, modulating vessel formation in normal tissues such as mouse ear and chicken chorioallantoic membrane (CAM), or inducing vessel growth in normally avascular tissues such as the cornea (Jain et al. 1997). The latter assay has an advantage over other in vivo assays in that the cornea is initially avascular. This model was originally developed in the rabbit eye, but has recently evolved by changing species to the smaller, less expensive, and genetically tractable mouse (Kenyon et al. 1996). The limitations of this assay, however, include the technically demanding nature of the surgery. Current models of in vivo evaluation of tumor angiogenesis generally consist of xenograft implantation of human cancer cell lines into immunodeficient mice. However, these tumor cells represent an extreme deviation from advanced cancers in vivo, and are not associated with the tumor stroma, which is a crucial component in tumor angiogenesis and subsequent metastasis (Bhowmick et al. 2004). Generally, xenograft models have limited ability to predict clinical efficacy of antiangiogenic anti-cancer agents. Tumor explants and three-dimensional models allow cell:cell and cell: matrix interactions to be examined in live tissue and cells, with endpoints including the measurement of angiogenic gene and protein expression as well as image analysis. More recently orthotopic xenograft of human tumors into nude mice has provided the ability to reproduce
both the histology as well as the angiogenic and metastatic pattern of most human cancers at advanced stage (Cespedes et al. 2006). While not useful in examining the contribution of the immune system in this process, this model is more promising than commonly used subcutaneous (SC) xenografts in preclinical drug screening and development. This model allows the evaluation of therapies in individual human tumors derived from different genetic backgrounds, as opposed to the use of inbred animals with a homogeneous genetic background (Cespedes et al. 2006). However, the ability of this model to predict clinical therapeutic response remains to be established. It is widely accepted that in vivo models are critical for defining the mechanism of drug activity and for testing therapeutic regimes. However, only a few models are ideal for this purpose. Zebrafish have become one of the most powerful and versatile models used for biomedical research to identify novel cancer markers/molecular signatures of disease (Amatruda et al. 2002). They are advantageous for drug screening as they account for the complex metabolism that affects drug efficacy or causes toxicity. Since chemicals can be directly delivered into the fish water and proteins can be injected, assessment of cytotoxic, apoptotic or anti-angiogenic effects of potential drug candidates singly, or in combination, is easy and straightforward (Parng et al. 2002; Kari et al. 2007). The transparency of zebrafish also allows direct imaging of mechanisms of cancer progression including cell invasion, intra-/extravasation, and angiogenesis (Stoletov et al. 2007; White et al. 2008). The development of xenograft zebrafish models has allowed the propagation and visualization of human cancer cells engrafted into optically transparent zebrafish (Marques et al. 2009). To date, zebrafish are the only animal organism to date with the potential for large scale, yet cost effective pharmacological screens to identify potential therapies to alter tumor dissemination and/or angiogenesis (Parng et al. 2002). Our group is using a zebrafish model to assess the anti-angiogenic properties of inhibitors of the TXS signaling pathway in vivo. While the effect of TXS pathway inhibitors in tumorigenesis has been investigated by our group and others (Jantke et al. 2004; Moussa et al. 2005; Sakai et al. 2006; Cathcart et al. 2011), the anti-angiogenic properties of these inhibitors are not fully understood. Using a transgenic line of zebrafish Tg (fli1:EGFP), which specifically expresses green fluorescent protein in vessels; we examined the effect of TXS-pathway inhibition on the morphology of intersegmental vessels (Fig. 5A). Selective TXS inhibition with ozagrel (10 µM) demonstrated clear effects on intersegmental vessel morphology following only 48 hour incubation (Fig. 5B), suggesting an anti-angiogenic mechanism for this drug. Similar anti-angiogenic effects on intersegmental vessel formation were observed following treatment with both a dual TXS/ EP4 inhibitor and a dual TXS/5-LOX inhibitor (Fig. 5B). These observations support the hypothesis that a dual targeting approach may further enhance the anti-angiogenic potential of single agent targeting alone. These fish can also be used to study the biology and pharmacology of human tumor in
Figure 5 Zebrafish is a pharmacological screening tool to identify novel anticancer therapies. A transgenic line of zebrafish Tg (fli1:EGFP), which specifically expresses green fluorescent protein in vessels may be used to examine the effect of potential anti-cancer compounds on intersegmental vessel morphology. Zebrafish are mated and eggs collected for screening. Approximately 6 h post-fertilization, developing larvae are added to culture plates (5 per well in duplicate). Screening compounds may be added directly to the zebrafish water for up to 4-days. Imaging and quantification of vessels is carried out by fluorescent microscopy (A). Selective TXS inhibition with ozagrel (10 µM) demonstrated clear effects on intersegmental vessel morphology following only 48 h incubation (B), suggesting an anti-angiogenic mechanism for this drug. Similar effects on intersegmental vessel formation were observed following TP antagonism, dual TXS/EP4 inhibition, and dual TXS/5-LOX inhibition. The morphological differences observed with dual TXS/EP4 inhibitor suggest potential developmental abnormalities and would suggest a titration of the drug is warranted.
