Evaluation of the Potential Application 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 [1]. In a previous study, a straightforward approach to produce injectable and in situ forming enzymatically cross-linked silk fibroin (SF) hydrogel was described [2]. The SF hydrogel, which has superior biocompatibility and fast-gelation property, 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 the SF hydrogel for inducing cancer cells apoptosis mediated by conformation transition, using one of the classical assays for studying angiogenesis in vivo, the chick embryo chorioallantoic membrane (CAM) assay [3]. Human cervical adenocarcinoma (HeLa) cells were encapsulated within the SF hydrogel and cultured for 4 days before being implanted in the CAM at day 10 of embryonic development. Acellular hydrogel and HeLa cells were used as controls. At the end of the assay (day 14), CAM excisions were obtained. Haematoxylin and eosin (H&E), SNA-lectin and Ki67 staining were performed in order to investigate possible inflammation, endothelial cells ingrowths and human proliferating cells presence. Cell apoptosis was detected 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 negative for Ki67 staining. It was possible to observe in the acellular sample that the 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 the 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.