1. Combined effect of charged particles irradiation and anticancer drugs in cultured tumor cells.(Milano and Roma 3 RDH_IRPT WP2) Combined radiochemotherapy treatment modality: -to improve locoregional tumour control and to reduce distant failure; -to reduce the total treatment Dose in radiosensitive patients In the last years: -several new drugs (various mechanisms of interaction with radiation) -many preclinical studies on the combined effect of standard radiotherapy and chemotherapy but very few data on the interaction of drugs with charged particles irradiation, a very promising treatment for many tumors due to the dosimetric and radiobiological properties. Milano and Roma 3, in collaboration with CNAO and Istituto Tumori - Milano, started studying the combined effect of charged particles (protons and C ions) or photon irradiation and anticancer drugs of new generation (Epothilone B and TTARHPS4), using cultured human tumor cells.

2. Measurements of dose-clonogenic survival curves of various tumor cells irradiated - at CNAO with a) protons and b) Carbon-ions - at Istituto Tumori with photons, combined or not with Epothilone B 1. Milano: Epothilone B, Microtubule Stabilizing Agent (MSA) : 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 .Radiosensitizer ?? Epothilone B reduces DNA repair capability of tumor cells. Epothilone B has shown antivascular and antiangiogenic effects and the ability to inhibit cell migration at non- cytotoxic concentration. At the present used for chemotherapic treatments : fewer side effects, superior pharmacological and anticancer activity, compared with previous MSA (taxanes). A promising agent for brain malignancies ( it is able to cross the blood-barrier )

3. Tumor 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) Equisurvival (  40% ) Epothilone B concentrations used: 0.125 nM U251 MG 0.075 nM A549 0,035 nM Daoy Invasive capacity, transwell invasion assay., (QCM ECMatrixassay kit-24-well- Merk Millipore,8um) Epothilone B treated cells ( compared to untreated ones ) : A549 55% U 251 MG 68%  Epothilone B riduce l’invasività di cellule A549 e U251

4. Survival of U251 MG, A549 and Daoy cells after (Protons +/- Epothilone B) treatment For all the cell lines Epothilone B increased protons (and photons, data not shown) cytotoxicity and the effect was more than additive. ( blu and green curves show calculated survival values for two additive interaction modalities: independent and overlapping ( addition of Epothilone B equivalent to an additional radiation Dose D*). Preliminary results reported at SIRR(2014) and at SIF ( 2015).

5. U251MG cells, protons and photons (4 independent exps) RBE 10% = 1.4 +/- 0.2 A549 cells, protons and photons (4 independent exps) RBE 10% = 1.5 +/- 0.2 Protons RBE ( 15 cm depth SOBP12_18 cm) DAOY cells, protons and photons (4 and 3 independent exps) RBE 10% = 1.2 +/- 0.1 Protons RBE depends on the cell line and…it is Not always 1.1 ! Next future ( 2016) : Interaction of Epothilone B and C ions and C ions RBE

6. Ora e prossimo futuro: Esperimenti e analisi dei risultati per le tre linee cellulari esposte a protoni e fotoni con e senza Epothylone B sono quasi conclusi Un lavoro con i risultati per U251 e A549 è stato inviato per pubblicazione. In fase di completamento l’analisi per le Daoy. Questa settimana inizieranno gli esperimenti con ioni C e cellule U251 dove, parallelemente alla sopravvivenza clonogenica si misurerà anche l’invasività cellulare dopo esposizione a ioni C con e senza Epothylone B. -Oltre agli effetti di Epothylone B, si determinerà anche RBE degli ioni C Misure citofluorimetriche