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

o Conventional radiotherapy bremsstrahlung photon beams from LINAC

o Hadrotherapy charged particles accelerated with syncrotrones or cyclotrones

Absorbed Dose:

For charged particles

Bethe - Block:

To irradiate the whole tumor thickness:

Spread Out Bragg Peak

(S.O.B.P.)

To irradiate each tumor section +

adeguate margins:

Pencil beam technology

Highly conformed dose distribution to the tumor

tumorsection

SUITABLE FOR

TUMORS

Deep seated

Close to critical organs

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 SYSTEMEstablished 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:

o Lung adenocarcinoma (A549)o Glioblastoma (U251MG)o Pediatric Medulloblastoma (DAOY)

MOST IMPORTANT BIOLOGICAL EFFECT 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)

24 h

Irradiation at ½ S.O.B.P.

(dose range = 0÷5 Gy)

Incubation37°C, 5% CO2, 90% umidity)

15 days

Clonogenic survival

vs. Radiation

dose/type +/- Epothilone B

MEASUREMENTS WITH PROTON BEAMS

Samples irradiated @ CNAO in Pavia

+ Epothilone B

Determination of Epothilone B concentration for each cell line

PRELIMINARY MEASUREMENTS

Similar to the concentration in

patient’s plasma

Equitoxic (survival ≈ 40%)for the various cell lines:

A2549 0.075 nMDAOY 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)

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)

DAOY (medulloblastoma) cells (4 indipendent experiments)

D.E.F.2Gy = 1.5

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

LamLam 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

INTERACTION BETWEEN RADIATION & EPOTHILONE B

ADDITIVITY?

SYNERGISM?ANTAGONISM?

A549 cells DAOYcells

LUTTJEBOER ANALYSIS

Synergism!

sinergism sinergis

m

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

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

NEXT FUTURE

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*.