Combined radiochemotherapy treatment modality:

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. 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 ) 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

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 primarymalignant 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 nMDaoy 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% EpothiloneB riducel’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 Protons RBE ( 15 cm depth SOBP12_18 cm) DAOY cells, protons and photons (4 and 3 independent exps) RBE 10% = 1.2 +/- 0.1 A549 cells, protons and photons (4 independent exps) RBE 10% = 1.5 +/- 0.2 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