In Vivo Evaluation of Silk Fibroin Hydrogel as Suppressor of Angiogenesis and Tumor Progression

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion and metastasis formation. Thus, the therapeutic suppression of angiogenesis could be the key to prevent tumor progression (Liu et al. 2007). In a previous study, an in situ forming enzymatically cross-linked silk fibroin (SF) hydrogel was prepared (Yan et al. National Patent Nr. 106041, 2011). The SF hydrogel undergoes a β-sheet conformation transition in vitro and in vivo that might be responsible for inhibiting encapsulated cells viability and proliferation. In this work, our aim was to investigate the potential application of SF hydrogel for inducing cancer cells apoptosis mediated by conformation transition, using the in vivo chick embryo chorioallantoic membrane (CAM) assay (Silva-Correia et al. 2012). Human cervical adenocarcinoma (HeLa) cells were encapsulated within the SF hydrogel and cultured before being implanted in the CAM. Acellular hydrogel and HeLa cells were used as controls. At the end of the assay, CAM excisions were obtained. Haematoxylin and eosin, SNA-lectin and Ki67 staining were performed in order to detect possible inflammation, endothelial cells ingrowths and human proliferating cells. Cell apoptosis was investigated by fluorescence microscopy by using the TUNEL assay. From staining observation, only a few small cell clusters were detected inside the cell-loaded hydrogel, which were negative for SNA-lectin and Ki67 staining. It was possible to observe that the acellular SF hydrogel does not allow endothelial cells infiltration and vessel formation. Some apoptotic cells were detected within the cell-loaded hydrogel, whereas no positive staining was observed in the acellular explants. This study demonstrates that SF hydrogel do not allow an effective formation of a solid tumor after β-sheet conformation transition, and thus it can be a very useful and tunable system for different biomedical applications, including suppressing angiogenesis and tumors progression in vivo.