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 24 april 2015

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 24 april 2015

3. Determination of Epothilone B concentration for each cell line 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% humidity) 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 (4 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 ) RDH_IRPT Meeting Roma 24 april 2015

6. U251MG cells, photons +/- Epothilone B (0.125 nM) (4 independent exps)

7. A549 cells, protons +/- Epothilone B (4 independent exps) RDH_IRPT Meeting Roma 24 april 2015

8. A549 cells, 6 MV photons +/- Epothilone B (4 independent exps)

9. DAOY cells, protons +/- Epothilone B (4 independent exps) RDH_IRPT Meeting Roma 24 april 2015

10. Measurements with photons to be done DAOY 2 independent exps 1-2 more exps to be performed preliminary results similar to those of protons RDH_IRPT Meeting Roma 24 april 2015

11. U251MG cells, protons and photons (4 independent exps) Protons RBE : RBE 2Gy,  = 1.5 +/- 0.2 RBE 10% = 1.5 +/- 0.2 A549 cells, protons and photons (4 independent exps) Protons RBE : RBE 2Gy,g = 1.5 +/- 0.2 RBE 10% = 1.4 +/- 0.2

12. 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 24 h Daoy A549 U251MG RDH_IRPT Meeting Roma 17 december 2014

13. To complete experiments with photons Carbon Ions ( 12 C 6+ ) +/- Epothilone B 3 cell lines Citofluorimetric analysis to measure Epothilone B effects on cells cycle 3 cell lines Epothilone B toxicity in cells from normal tissues Next Future