Development of in vitro models of invasion for the pharmacological investigation of small molecule inhibitors of tumour progression. Development and validation of a 3-dimensional tumour spheroid invasion model to evaluate the pharmacological effects of novel small molecule β3 integrin antagonists.

摘要

Tumour dissemination is a major reason for failure of therapy for many tumour types therefore there is a requirement for novel targets & therapies. The αIIbβ3 and αvβ3 integrins have been demonstrated to have significant involvement at many stages of the tumour dissemination process including, tumour cell adhesion, migration, metastasis and angiogenesis, and thus the β3 integrins are a potential target for therapeutic antagonism with small molecules. Because of the clear interaction between the different integrin types, targeting integrins as a therapeutic strategy requires targeting more than one integrin type. Consequently, the ICT is developing a group of novel new αIIbβ3 and αvβ3 integrin dual antagonists. One of the main challenges is having a relevant, validated experimental model that expresses these integrins. The aim of the work presented here is to develop and validate an in vitro αIIbβ3 and αvβ3 integrin expressing assay of tumour cell invasion. The spheroid invasion assay has the advantage over standard monolayer transwell chamber invasion assays of being a 3-dimensional assay, and thus mimics better the cell-cell interactions and architecture that are present in a tumour compared to the monolayer-based assay. A panel of human cancer cell lines known to express one of the molecular targets of interest, αvβ3 integrin was evaluated for the ability to form spheroids and to invade through collagen matrices. One glioma cell line, U87-MG, demonstrated consistent spheroid formation and invasion and was thus selected for further studies. Optimum conditions were established for use of U87-MG in the invasion assay, and the assay was validated using a known inhibitor of invasion, LiCl and known β3 antagonist, cRGDfV. Subsequently a group of novel small molecule β3 antagonists were evaluated at nontoxic concentrations using the assay. Both LiCl and cRGDfV inhibited spheroid invasion through the gel in a dose-dependent manner, thus validating the assay. Furthermore, when the novel small molecule β3 antagonists were evaluated using the model, a dose and time dependent reduction in U87-MG spheroids invasion in collagen was observed. In further work initial steps were taken to construct a cell line which expresses both αIIbβ3 and αvβ3 integrin to use in the model to assess for dual integrin antagonism. In conclusion, this work has established a validated assay which has been utilised for some compounds to evaluate a group of novel small molecule β3 integrin antagonists with encouraging results.