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

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

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

4. 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

5. 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

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

7. 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

8. 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)

9. 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)

10. Measurements with proton beams
Samples 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

11. 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  nM
DAOY  nM
U251MG  nM
Clonogenic survival of A549 cells vs. Epothilone B concentration

12. 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:

13. 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

14. 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 , 1979
G. Steel, Int. J. Radiation Oncology Biol. Phys. , V. 5, pp , 1979
Lam
Lam G. K. Y., Bull. Math. Biol. Vol. 51 pp , 1989
Luttjeboer
Luttjeboer M. et al., Int. J. Rad. Biol. Vol 86, pp , 2010

15. Luttjeboer Analysis Synergism! A549 cells DAOYcells sinergism

16. 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

17. 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

18. Thank you!

20. 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

21. 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!

22. 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*.