Combinazione di bevacizumab e radioterapia nel melanoma uveale: uno studio in vitro

Uveal melanoma (UM) is the most common tumour of the eye with an incidence of 5.5-10.9 cases per million people per year. It frequently results in a reduced eye function and in 50% of patients gives rise to hepatic metastases that inevitably lead to death. The first choice treatment for UM is radiotherapy (RT), which can be associated to an anti-angiogenic agent, in order to prevent an increase of VEGF level in ocular fluids and radiation side effects like retinopathy. UM is a highly vascularised tumour therefore an anti-angiogenic treatment may enhance its response to RT both by normalising blood vessels and improving tissue oxygenation, as reported in several studies for variuos types of cancer. A recent preclinical study from Sudaka et al. (2013) has shown an additive effect of the anti-VEGF drug bevacizumab and RT on mice tumours induced by inoculation of human uveal melanoma cells OCM-1. A similar additive effect was observed in vitro on the viability of OCM-1 cells co-cultured with human umbilical vein endothelial cells (HUVEC) in a transwell plate. In vitro, effects due to vessel regression do not bias results, therefore this project was designed to investigate if bevacizumab may exert an alternative action on tumour microenvironment that can be responsible for the above described results. Firstly we showed that in vitro VEGF pathway is activated since OCM-1 cells release VEGF in the culture medium, and both OCM-1 and HUVEC cells express VEGFR2, even when they are grown separately. Secondly we demonstrated that in transwell co-cultures pre-treating cells with bevacizumab prevents radiation-induced increase in VEGF levels, similarly to what happens in vivo. The inhibition of the VEGF pathway has a direct anti-proliferative effect on several cancer cell types, including melanoma, that express VEGF receptors, and it can potentially affect their ability to survive radiation treatment. Thus we tested if the decrease in VEGF concentration caused by bevacizumab directly affected OCM-1 cell radiosensitivity. Our results showed that, although bevacizumab at high concentration causes a moderate decrease in the survival fraction of OCM-1 cells grown as a monoculture, it does not enhance their response to radiation. Therefore we hypothesised that the radiosensitizing effect of bevacizumab on co-cultured UM cells may depend on a crosstalk between cancer and endothelial cells. IL-8 is frequently coexpressed with VEGF in tumours where they are reciprocally upregulated and cooperate to induce angiogenesis . In addition it can act as a growth factor for cancer cells and as a pro-angiogenic mediator at the same time. For these reasons and since its concentration is generally high in ocular fluids of UM patients, we analysed the role of the chemokine in the UM-endothelium microenvironment. We found that both OCM-1 and HUVEC cells constitutively express IL-8 and its receptor CXCR2. Moreover, in both cell types we observed an inhibitory effect of bevacizumab on IL-8 release, associated to a lower reduction in cell viability. The drug also caused a marked decreased of CXCR2 expression in OCM-1 cells. Altogether these results suggest that one of the mechanisms by which bevacizumab improves UM cell response to radiation may be the downregulation of IL-8 pathway in the tumour macroenvironment. As a future perspective it would be interesting to determine intratumoral IL-8 levels in vivo and IL-8 concentration in transwell plate co-cultures, in order to better understand how they vary in response to anti-angiogenic agents, RT, or they combination.