ESTRO 36 Abstract Book

S546 ESTRO 36 _______________________________________________________________________________________________

4 Candiolo Cancer Institute- Torino- Italy, Medical Physics Unit, Candiolo TO, Italy Purpose or Objective The important goal of tumor immunotherapy is to identify strategies to modulate in vivo the anti-tumor immunity in order to achieve clinical efficacy. In the last decade great progresses have been made in the field of tumor immunology and an increasing number of tumor antigens have been identified. In particular, several different engineering T-cells were designed to express receptors specific for antigens expressed in the tumor compartment. Herein, in order to block tumor progression, we employed Chimeric Antigen Receptors (CARs) technology to target the tumor vasculature. To this aim we used a spontaneous mouse tumor model of pancreatic neuroendocrine insulinoma, RIP-Tag2. Despite its low frequency in cancer patients, we chose this model, since develops invasive tumors through well-characterized and synchronous pre- malignant stages, sustained by active angiogenesis. Little is known about the effects of ionizing radiation on tumor burden of RIP-Tag2 mice and the impact of whole-body irradiation on the overall survival. Material and Methods In the current study tumor-bearing RIP-Tag2 mice were irradiated with two different sub-lethal dosages (5 and 6 Gy respectively), by means of 6MV x rays of Tomotherapy; a treatment plan was performed and evaluated for each cage pie . We hypothesized that sub-lethal radiation might be more effective than a lethal dose radiation and clinically acceptable in promoting anti-tumor immunity. The day after the irradiation mice were injected with enginered T-cells (20 million cells). Results Firstly,we observed that irradiation per se did not affect tumor growth and all the mice survived until the end of treatment. Furthermore, by means of this approach, we found statistically significant inhibition of tumor growth in mice treated with anti-vasular CAR-T-cells compared with controls. Interestingly, we noticed a strong reduction in tumor vessel area and a decrease of blood vessel permeability. Conclusion These data suggest that the tumor vasculature can be efficiently targeted by specific CAR-T-cells, causing a significant reduction in the tumor burden of RIP-Tag2 mice and potentially in other tumor types. PO-0990 Combining radiotherapy and notch inhibition in melanoma K. Thippu Jayaprakash 1,2 , M. Hussein 3 , A. Nisbet 3,4 , R. Shaffer 2 , M. Ajaz 1,2 1 University of Surrey, Department of Clinical and Experimental Medicine, Guildford, United Kingdom 2 St Luke's Cancer Centre- Royal Surrey County Hospital, Department of Oncology, Guildford, United Kingdom 3 St Luke's Cancer Centre- Royal Surrey County Hospital, Department of Medical Physics, Guildford, United Kingdom 4 University of Surrey, Department of Physics, Guildford, United Kingdom Purpose or Objective Melanoma is classically viewed as a radioresistant tumour. Phenotypic plasticity, specifically the emergence of a cancer stem cell (CSC) population, may be one reason for this. The notch signalling pathway plays a crucial role in maintenance of the CSC phenotype, and also in cell migration. This pathway is frequently aberrant in melanoma and is therefore a potential mechanism for the observed radioresistance. The aims of this project were (1) to investigate whether notch inhibition with the γ - secretase inhibitor, RO4929097 that targets γ - secretase cleavage and thereby inhibiting the notch signalling pathway, improves the radiation sensitivity of melanoma

cell lines; (2) to investigate the effects of notch inhibition and radiotherapy on melanoma cell migration. Material and Methods Two melanoma cell lines, A375 and SKMEL28 were irradiated with 250 kV x-rays to 1, 2, 4, 6, 8 and16 Gy in combination with 1, 3, 10, 30 and 100 µm of RO4929097 in 96-well plates. Cells were permitted to grow for a further 5 - 7 days and viability was assessed with the MTS assay. Loewe’s combination index (CI) was used (CI=(C A , X /IC X, A )+(C B , X /IC X, B )) to evaluate the interaction between radiation and RO4929097. For cell migration experiments, A375 and SKMEL28 cells were treated with 10 and 100 µm of RO4929097, alone and in combination with radiation (2 and 8 Gy) in 6-well plates. A scratch was performed and daily light field microscope photographs were taken. In all experiments, radiation was delivered one hour after cells were treated with RO4929097. Results Loewe’s CI of < 1 and > 1 are taken to indicate synergism and antagonism respectively. The Loewe’s combination index analysis reproducibly showed strong synergy in A375 melanoma cells when radiation doses of 1, 2, 4, 6 and 8 Gy were combined with 100 µm of RO4929097 and a trend towards mild synergy was observed with lower doses of radiation and higher doses of RO4929097 (Figure 1 ). This may be due to a reduction in the number of CSCs by RO4929097 that renders lower radiation doses more effective. Similar patterns of interaction were observed for SKMEL28 cells.

Cell migration assays showed that cell migration was inhibited in both cell lines following treatment with 10 and 100 µm of RO4929097 and this was more pronounced at 100 µm, and similar effects were seen when radiation was combined with RO4929097. 8 Gy alone failed to control cellular migration but this was abrogated by the addition of RO4929097 (Figure 2) .

Conclusion Inhibition of the notch signalling pathway increases the radiosensitivity of melanoma cells. We hypothesise this is