A new screening platform for fluorescence imaging probes based on the chick embryo chorioallantoic membrane (CAM)

This thesis aims to validate the Chicken chorioallantoic membrane (CAM) platform as an alternative to animal experiments which are usually performed for evaluating the efficacy of diagnostic probes. The choice to utilize the CAM branches from the fact that the model provides a rapid and cost-effective platform, allowing direct visualization of tumor development and thus making it suitable for different applications, such as in vivo imaging. Additionally, it represents an innovative approach from a sustainability perspective and would reduce the use of animals, at least in the initial screening phase. Here, the focus has been on the application of CAMs for the initial screening of probes for fluorescence cancer imaging in human cell lines. The study was performed with the fluorescent probe RGD-800, targeting integrins, which are highly expressed in certain human tumoral cell lines. In particular, RGD-800 is composed of the fluorophore IRDye800CW linked to an RGD targeting moiety. In vitro tests revealed RGD-800 higher affinity for αvβ5 and αvβ3 proteins with respect to αvβ6. Additionally, experiments assessing the cellular uptake by flow cytometry demonstrated that RGD-800 bound specifically to integrins expressed on the surface of U-87 MG human glioblastoma cells, with decreased binding in presence of an excess of competitor and accumulation into the cells in a time-dependent manner. When a blocking solution of RGD was co-injected with RGD-800 in CAMs bearing U-87 MG xenografts, a significant reduction in fluorescent signal was observed. Ex vivo fluorescence quantification and western blot analysis confirmed the observed signal reduction and the integrin expression in the tumor xenografts, respectively. These experiments proved that the CAM platform could be used to test the specificity of the probes to their targeted receptors, in this case RGD-800 binding to αvβ5 and αvβ3 integrins. The thesis aims to demonstrate the advantages and difficulties of the CAM model, as well as to explore alternative methods that can be used to overcome them, ultimately demonstrating that the in ovo platform represents a valid and effective alternative to traditional animal testing. Moreover, the results imply that the RGD-800 probe could serve as a valuable tool for imaging applications, such as fluorescence-guided surgery.

This thesis aims to validate the chicken chorioallantoic membrane (CAM) platform as an alternative to animal experiments which are usually performed for evaluating the efficacy of diagnostic probes. The choice to utilize the CAM branches from the fact that the model provides a rapid and cost-effective platform, allowing direct visualization of tumor development and thus making it suitable for different applications, such as in vivo imaging. Additionally, it represents an innovative approach from a sustainability perspective and would reduce the use of animals, at least in the initial screening phase. Here, the focus has been on the application of CAMs for the initial screening of probes for fluorescence cancer imaging in human cell lines. The study was performed with the fluorescent probe RGD-800, targeting integrins, which are highly expressed in certain human tumoral cell lines. In particular, RGD-800 is composed of the fluorophore IRDye800CW linked to a RGD targeting moiety. In vitro tests revealed RGD-800 higher affinity for αvβ5 and αvβ3 proteins with respect to αvβ6. Additionally, experiments assessing the cellular uptake by flow cytometry demonstrated that RGD-800 bound specifically to integrins expressed on the surface of U-87 MG human glioblastoma cells, with decreased binding in presence of an excess of competitor and accumulation into the cells in a time-dependent manner. When a blocking solution of RGD was co-injected with RGD-800 in CAMs bearing U-87 MG xenografts, a significant reduction in fluorescent signal was observed. Ex vivo fluorescence quantification and western blot analysis confirmed the observed signal reduction and the integrin expression in the tumor xenografts, respectively. These experiments proved that the CAM platform could be used to test the specificity of the probes to their targeted receptors, in this case RGD-800 binding to αvβ5 and αvβ3 integrins. The thesis aims to demonstrate the advantages and difficulties of the CAM model, as well as to explore alternative methods that can be used to overcome them, ultimately demonstrating that the in ovo platform represents a valid and effective alternative to traditional animal testing. Moreover, the results imply that the RGD-800 probe could serve as a valuable tool for imaging applications, such as fluorescence-guided surgery.