Protocol development for discovery of angiogenesis inhibitors via automated methods using zebrafish

Their optical clarity as larvae and embryos, small size, and high fecundity make zebrafish ideal for whole animal high throughput screening. A high-throughput drug discovery platform (HTP) has been built to perform fully automated screens of compound libraries with zebrafish embryos. A Tg(kdrl:EGFP) line, marking endothelial cell cytoplasm, was used in this work to help develop protocols and functional algorithms for the system, with the intent of screening for angiogenesis inhibitors. Indirubin 3’ Monoxime (I3M), a known angiogenesis inhibitor, was used at various concentrations to validate the protocols. Consistent with previous studies, a dose dependant inhibitory effect of I3M on angiogenesis was confirmed. The methods and protocols developed here could significantly increase the throughput of drug screens, while limiting human errors. These methods are expected to facilitate the discovery of novel anti-angiogenesis compounds and can be adapted for many other applications in which samples have a good fluorescent signal.

Pathological angiogenesis occurs with a deregulation in homeostasis. In disorders 94 featuring excessive angiogenesis, there is a surplus of angiogenesis activators that may be 95 accompanied by the suppression or reduction of angiogenesis inhibitors. Cancer, one of the 96 leading causes of death worldwide, is the most prominent disease in this category. Tumour 97 angiogenesis has been extensively studied to characterize its mechanisms and to develop targeted 98 and more effective therapies. Although, tumors come in many forms, angiogenesis is a process 99 to which all tumor progression is dependent. Two distinguishing features of cancer, 100 uncontrollable cell growth and metastasis, cannot be sustained in the absence of 101 neovascularization [7].

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Inhibition of a pro-angiogenesis pathway, such as VEGF signaling, can block 103 angiogenesis in tumors and also change or destroy existing tumor vessels [8]. VEGF inhibitors 104 are effective in several types of cancers, however, the benefits are transient, and the vast majority 105 of patients who initially respond to the therapies develop resistance over time [9]. This indicates 106 the existence of alternate pathways for cancer angiogenesis.

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There have been cases in which VEGF inhibitors were found to be more effective by 108 targeting multiple pro-angiogenesis pathways. Sunitinib, a drug approved for the treatment of 109 renal cell carcinoma (RCC) and imatinib-resistant gastrointestina stromal tumor (GIST), is an 110 example. This compound is a receptor tyrosine kinase (RTK) inhibitor that inhibits all VEGF 111 and PDGF receptors, which are upregulated in clear cell RCC [10]. Thus, it is possible to tailor 112 treatments towards specific cancers with VEGF inhibitors that also inhibit alternate pathways of 113 angiogenesis.

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This report presents the development and validation of methods that will be used to  Instruments] to house compound plates. Figure 1 (Fig 1) shows a top view of a 3D CAD 138 drawing of the HTP.   counts when compared to the control group, as shown in Figure 4 (Fig 4). I3M was shown to pathways, much like in human tumors [16]. This together with its high fecundity makes it ideal 9 222 for statistically significant whole animal high throughput screening for angiogenesis inhibitors.

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The HTP provides a powerful and efficient tool for the discovery of drugs that modulate 224 angiogenesis.

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It is worthy to note that, in addition to the described methods, certain interventions were 226 implemented to circumvent some limitations of working with the HTP and zebrafish. When were found to be more effective by also targeting other angiogenesis pathways in certain cancers 250 [10]. The use of these methods is expected to result in the discovery of potentially novel anti-251 angiogenesis compounds that may improve treatment regimens for cancer.

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The methods presented are from the perspective of a search for anti-angiogenesis sample cup had to be limited to avoid having multiple embryos dispensed at once. The average 284 specific gravity of the embryos did not allow them to rise above a certain level while they were 285 mixed with the systems magnetic stir bar. For this reason, the optimal embryo to E2 water levels 286 were 100 embryos for a water level at 1.8 cm above the sample cup base, 200 embryos for water 12 287 levels between 1.8 cm and 3.2 cm, and 300 embryos for levels above 3.2 cm. Adding more than 288 300 embryos would result in dispensing errors. In addition, a vigorous trial and error process 289 was used to find the optimal settings to dispense 1 embryo/well with 40 µL of E2 embryo 290 medium. The pressure parameters can be found in Figure 6 (Fig 6). All other settings can be 291 found in Supplementary Figure 2 (S2 Fig). Supplementary Video 1 (S1 Vid) shows the COPAS 292 in the process of dispensing 1 embryo/well in a 96-well plate. The markings at 1.8 cm and 3.2 293 cm above the sample cup base can also be observed in the video.  The Sciclone was also used to dispense clove oil from a reservoir container into the 326 sample plate. The program used for this is identical the one described for dispensing compounds 327 into the sample plate from the compound plate, with one difference being that the tips descend 328 into a reservoir container at 0.2 mm above its bottom for aspiration instead of 2 mm above the The data generated and/or analysed during this work, if not already included in this manuscript, 367 can be obtained from the corresponding author upon request.