###### Abstract
There is only scarce data regarding the response of human melanoma cell lines along with the proton spread-out Bragg peak. According to these data, at the distal end of a 65 MeV proton SOBP, an RBE of 1.27 and even higher for the more resistant melanoma cell lines were recorded [17; 18; 19].More recently, work carried out on uveal melanoma Mel270 cells at the mid-SOBP position of a clinical high-energy proton beam showed an increased anti-migratory effect compared to photon irradiation [20]. Currently, many commercial Treatment Planning Systems (TPSs) for proton therapy incorporate the capability of calculating LET distributions and RBE estimations, but there remain challenges on how to deal with uncertainties resulting from potential RBE variations. Future TPS that would potentially be able to take into account RBE variability, could reduce the toxicity and the incidence of later-occurring inducedorbidities, thus making it possible to exploit the full potential of proton therapy [2]. The main aim of this work was to study the RBE values at the clinical SOBP of the CATANA proton therapy facility. Radiation-induced biological effects measured in terms of clonogenic cell death (see below) were predicted by the LEM II (Local Effect Model) [21], Monte Carlo approaches [22], and semi-empirical models based on LET experimental measurements [23]. All adopted methods were then compared with in-vitro experimental results. These were evaluated using the human 92.1 uveal melanoma cell line, a well-known model to investigate the aggressive behaviour of such types of neoplasia. In order to compare the effects induced by proton therapy in a non-tumorigenic cell line the human normal retinal pigment epithelial ARPE-19 cell line was also irradiated. The normal cells can easily recover from damage induced by ionizing radiation due to a more efficient damage response machinery with respect to the cancer cells. Moreover, choosing the normal retinal pigment epithelial ARPE-19 cell line allows obtaining insights into the magnitude of biological effects induced by an erroneous dose distribution during a proton therapy treatment plan. The retina is the organ at risk in uveal melanoma treatment and despite the usually excellent precision granted by protontherapy, the anatomical structure of the eye, with dimensions in the order of the millimeter is such that lack of a rigorous dose contouring is possible in actual clinical scenarios, leading to unwanted dose deposition. Such information is therefore of paramount importance to provide an estimate of the possible adverse effects deriving by beam forward straggling. Microdosimetry experimental spectra acquired to estimate the proton LET in different positions along the SOBP, were measured in the same experimental conditions of the biological sample irradiation. Two different detectors were used to this scope: a mini-TEPC [24; 25], developed at the Legnaro National Laboratories of the National Institutefor Nuclear Physics (LNL-INFN) and a Silicon On Insulator (SOI) microdosimeter with 3D Sensitive Volumes (MicroPlus-Bridge) [26; 27]. The Loncol's weighting function was then applied to assess the microdosimetric RBE and compare it with the experimental one. The biological damage was estimated also adopting a computational method which couples Geant4 with the LEM-II model as well as the pure LEM-II model. In such a way, it was possible to compare the well-established LEM with a Monte Carlo method and a semi-empirical one and to establish an approach that best reproduces the biological data at a depth of 24 mm, the mid-position of the spread-out Bragg peak.