1. WP2. Combined effect of charged particles irradiation and anticancer drugs in cultured human tumor cells (Milano and Roma 3, collaboration with CNAO and Istituto Tumori_Mi) ? to improve locoregional tumor control and to reduce distant failure for radioresistant and invasive tumors ? to reduce the total treatment Dose in radiosensitive patients RDH_IRPT Meeting Roma 17 december 2014

2. Epothilone B, Microtubule Stabilizing Agent (MSA) At the present used for chemotherapic treatments : fewer side effects, superior pharmacological and anticancer activity, compared with previous MSA (taxanes). Defective mitotic spindle formation, cell cycle arrest in M phase, apoptosis or post mitotic death. Accumulation of cells in the most radiosensitive G2-M phase of the cell cycle. Cell lines: A549 (Non-Small Cell Lung Cancer) very frequent tumor in adults, leading cause of cancer related death in Europe U251MG (glioblastoma) the most aggressive primary malignant brain tumor in adults DAOY (medulloblastoma) the most common malignant brain tumor of childhood End Point: Clonogenic survival and growth curves of the tumor cells irradiated with proton, carbon-ion and photon therapeutic beams, combined or not with Epothilone B RDH_IRPT Meeting Roma 17 december 2014

3. Determination of Epothilone B concentration for each cell line P RELIMINARY MEASUREMENTS Similar to the concentration in patient’s plasma For equitoxicity in the various cell lines: A2549  0.075 nM DAOY  0.035 nM U251MG  0.125 nM Clonogenic survival of A549 cells vs. Epothilone B concentration

4. 24 h Irradiation at ½ S.O.B.P. (dose range = 0÷5 Gy) Incubation 37°C, 5% CO 2, 90% umidity) 15 days Clonogenic survival vs. Radiation dose/type +/- Epothilone B MEASUREMENTS WITH PROTON BEAMS Samples irradiated with charged particles at CNAO in Pavia + Epothilone B

5. Results U251MG cells, protons +/- Epothilone B (3 independent exps) Interaction between protons and Epothilone B: Luttjeboer M. et al (2010) Soverl.( D rad + Chem.) = exp(-  D* -  D* 2 ) D * = (D equiv + D rad ) S( D equiv )=S(Chem) D equiv = 1.5 Gy S(Chem) = 0.38 Sindep.(D rad +Chem.) = S(Chem)xS( D rad ) Scombi  Soverl Synergism Scombi  Sindep Antagonism RDH_IRPT Meeting Roma 17 december 2014

6. Results A549 cells, protons +/- Epothilone B (4 independent exps) RDH_IRPT Meeting Roma 17 december 2014

7. Results DAOY cells, protons +/- Epothilone B (4 independent exps) RDH_IRPT Meeting Roma 17 december 2014

8. Conclusions up to now: These preliminary results seem to indicate that interaction between proton irradiation and Epothilone B is synergic for all the studied cell lines. RDH_IRPT Meeting Roma 17 december 2014

9. Measurements with photons A549 3 independent exps DAOY 2 independent exps U251MG 3 independent exps analysis completed, results similar to those of protons 1 more exp to be performed preliminary results similar to those of protons For 2 exps, data analysis is in progress RDH_IRPT Meeting Roma 17 december 2014

10. Epothilone B effects on cells cycle.. Just started :: Citofluorimetric analysis to measure Epothilone B effects on cells cycle. Data are shown for untreated cells In progress: analysis of samples treated with Epothilone B for 24 h and fixed at various times between 0 and 72 h Daoy A549 U251MG RDH_IRPT Meeting Roma 17 december 2014

11. To complete experiments with photons Carbon Ions ( 12 C 6+ ) +/- Epothilone B To complete Citofluorimetric analysis to measure Epothilone B effects on cells cycle Epothilone B toxicity in cells from normal tissues Next Future