Interaction between hadrons and chemotherapy agents in human cancer cells cultured in vitro First Year Workshop 2014 Miriam Lafiandra miriam.lafiandra@unimi.it Supervisor: Prof. P.F. Bortignon Co-Supervisor: Prof. D. Bettega

Hadrotherapy with charged particles Conventional radiotherapy  bremsstrahlung photon beams from LINAC Hadrotherapy  charged particles accelerated with syncrotrones or cyclotrones

For charged particles Bethe - Block: Absorbed Dose: For charged particles Bethe - Block:

Highly conformed dose distribution to the tumor To irradiate the whole tumor thickness: Spread Out Bragg Peak (S.O.B.P.) To irradiate each tumor section + adeguate margins: Pencil beam technology tumor section Highly conformed dose distribution to the tumor

Close to critical organs Resistant to conventional therapies Deep seated Close to critical organs Suitable for tumors Resistant to conventional therapies Dose distribution calculated for a medulloblastoma treatment planning

Chemoradiotherapy in RDH INFN project Therapeutic radiations (photons/hadrons) Chemical agents  radiosensitizing action aims: To increase tumor local control To reduce metastasis’ formation probability

Epothilone B Potential radiosensitizing agent: Interferring with cell’s cycle, it stops cells in G2/M (cells in this phase are very radiosensitive) It inhibits DNA repair mechanisms in tumor cells

Biological system Established human tumor cell-lines cultured in vitro: characteristics do not change over time (i.e. constant proliferative capacity) Experiments in controlled and reproducible conditions! Cell Lines now in study: Lung adenocarcinoma (A549) Glioblastoma (U251MG) Pediatric Medulloblastoma (DAOY)

Most important biological effect LOSS OF CLONOGENIC CAPACITY in radiotherapy: LOSS OF CLONOGENIC CAPACITY IN CANCER CELLS Survived cell after irradiation ↔ it generates a colony made up of at least 50 cells (5-6 cell divisions)

Measurements with proton beams Samples irradiated @ CNAO in Pavia + Epothilone B Irradiation at ½ S.O.B.P. (dose range = 0÷5 Gy) 24 h Incubation 37°C, 5% CO2, 90% umidity) Clonogenic survival vs. Radiation dose/type +/- Epothilone B 15 days

Preliminary measurements Determination of Epothilone B concentration for each cell line Similar to the concentration in patient’s plasma Equitoxic (survival ≈ 40%) for 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

Preliminary results with proton beams (1/2 sobp 15 cm) A549 (lung adenocarcinoma) cells (4 indipendent experiments) Surviving curves described by Linear Quadratic Model Protons α= (0,60 ± 0,07) Gy-1 β= (0,04 ± 0,02) Gy-2 Protons + Epothilone B α= (0,90 ± 0,04) Gy-1 S0= (0,36 ± 0,02) Dose Enhanchment Factor:

Preliminary results with proton beams (1/2 sobp 15 cm) DAOY (medulloblastoma) cells (4 indipendent experiments) Protons α= (0,57 ± 0,05) Gy-1 β= (0,03 ± 0,02) Gy-2 Protons + Epothilone B α= (0,88 ± 0,03) Gy-1 S0= (0,36 ± 0,03) D.E.F.2Gy = 1.5

Interaction between radiation & Epothilone B ADDITIVITY? ANTAGONISM? SYNERGISM? Various analysis based on different definitions of additivity between citotoxic agents: Steel & Peckham G. Steel, M. Peckham et al., Int. J. Radiation Oncology Biol. Phys. ,V. 5, pp 85- 91, 1979 G. Steel, Int. J. Radiation Oncology Biol. Phys. , V. 5, pp 1145-1150, 1979 Lam Lam G. K. Y., Bull. Math. Biol. Vol. 51 pp. 293-309, 1989 Luttjeboer Luttjeboer M. et al., Int. J. Rad. Biol. Vol 86, pp. 458-466, 2010

Luttjeboer Analysis Synergism! A549 cells DAOYcells sinergism

Work in progress comparison with photons beams: PRELIMINARY RESULTS  synergistic interaction between photon beams and Epothilone B more measurements on proton beams with A549, DAOY cells extension of these measurements to U251MG (glioblastoma) cells

Next Future Carbon Ions (12C6+) +/- Epothilone B : (greater biological effectiveness & better physical properties) Citofluorimetric analysis to measure Epothilone B effects on cells cycle Epothilone B toxicity in cells derived from normal tissues

Thank you!

Synergic interaction’s consequences Effect of the combined treatment is greater than the sum of the effects of radiation and chemoterapy used alone it is possible to reduce radiation dose to obtain the same effect of a standard treatment Combining the local action of radiation and the systemic one of the chemical agent  major effect inside tumor volume + reduction of metastasis’ formation probability. Epothilone B has a major effectiveness on highly proliferating cells (such as cancer cells)  selectivity

Linear quadratic model Hp: Letal DNA damages caused by radiation can be due to a single track damages frequency directly proportional to radiation dose. Letal damage can be even caused by the interaction of lesions due to different indipendent tracks  frequency quadratically proportional to dose. Letal lesions randomly distributed. Epothilone B combined with radiation makes the β parameter negligeble!

Steel & Peckham analysis Isoeffect plane For a given survival level S*: Mode I: for every chemoterapy agent’s concentration, it is reported the radiation doses that added to the chemical agent leeds to S*. Mode II: for every concentration, it is calcolated the dose needed to produce the same effect It is then reported the dose that added to this calculated one, leeds to S*.