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Lorenzo Manti and Francesca Margaret Perozziello

The radiobiology of laser-driven particle beams: focus on sub-lethal responses of normal human cells. Lorenzo Manti and Francesca Margaret Perozziello Physics Department, University of Naples Federico II & INFN Naples Section, Naples, Italy. The 3 rd ELIMED workshop

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Lorenzo Manti and Francesca Margaret Perozziello

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  1. The radiobiology of laser-driven particle beams: focus on sub-lethal responses of normal human cells Lorenzo Manti and Francesca Margaret Perozziello Physics Department, University of Naples Federico II & INFN Naples Section, Naples, Italy The 3rd ELIMED workshop MEDical and multidisciplinary applications of laser-driven ion beams at ELI-beamlines INFN-LNS, Catania, 7-10 September 2016

  2. Preamble • Laser-driven particles as probes of radiation action • Dromey et al., Picosecond metrology of laser-driven proton bursts, Nat Commun(2016) • Ultra-fast high-energy radiobiology • Malka et al., Ultra-short electron beams based spatio-temporal radiation biology and radiotherapy, Mutat Res (2010) • Alternative modality of hadrontherapy • Ledingham et al., Towards Laser Driven Hadron Cancer Radiotherapy: A Review of Progress ApplSci (2014)

  3. Spatio-temporal radiobiology

  4. Why radiobiology matters • Does laser-driven acceleration regime impact on cellular radioresponse?

  5. Radiation-induced DNA damage:temporal scale

  6. Low LET radiation biological action:chain of events  fast electron incident x-ray photon ion radical  chemical changes due to the breakage of bonds  biological /organismal effects

  7. Energy deposition by ionising radiation is highly inhomogeneous Charged particles are more effectivebecause of clusteringof ionizations within individual tracks • This simple Double-Strand • Break has been produced • by: • one direct ionization, and • one OH radical diffusing • from an ionization in water • very nearby • i.e. both were from a small • cluster of ionizations in a • single electron track • Other DSB can be due: • to two direct ionizations (ie Direct only) • or to two OH radicals (ie Indirect only) DSB result from clustering of ionizations on nm scale

  8. Two examples of Complex Clustered Damage in DNA Single tracks of ‘low’- LET or high- LET radiation can produce Complex Clustered Damage in DNA Parts of: Electron track Alpha-particle track

  9. 5.3 MeV alpha particle (1.34 MeV/u 4He ion) LET ~ 88 keV/µm By courtesy of HerwigParetzke, Werner Friedland and Maximilian Kriepl Track simulation methods in Kreipl et al, Radiat Environm Biophys 48, 349-359 (2009).

  10. The Oxygen Effect-1 Oxygen is the best known and most general radiation sensitizer. Dose(hypoxia) Dose(oxygenated) OER = OER usually about 3 at high radiation doses, but can be lower at low doses. (from Hall 2000)

  11. The oxygen effect-2 • Tumour hypoxia • Wardman “Time as a Variable in Radiation Biology: The Oxygen Effect, Radiat. Res (2015) • Hadrontherapy

  12. The Dose-Rate Effect • The proportion of cells killed by a given dose of x- or -rays is reduced if the dose delivery is protracted by: • Low dose-rate. • Dose fractionation • In both cases the reason is sub-lethal damage repair

  13. Number of Mutations/Locus x 105 Dose (R)

  14. Ultra-high dose rate • Doses of the order of thousand of cGymay be delivered in a small fraction of a second • Two types of machines. • Discharge machines store in a bank of condensers an enormous amount of electrical energy, which if discharged instantaneously produce a pulse of electrons lasting 3 to 50 ns. The electrons may be used directly to irradiated biological specimens or directed onto targets to convert the energy to a pulse of X-rays. Typical dose rates are 1011Gy/min. • Electron linear accelerators. The dose may be given in a single pulse lasting 1 ms, but in fact it is not delivered continuously throughout this pulse. Because of the frequency of the RF power supply, electrons are emitted largely during several thousands short periods during the pulse.The instantaneous dose-rate may again approximate to 1011Gy/min.

  15. Oxygen depletion effect • If both the dose and instantaneous dose rateare sufficiently high, the rapid deposition of radiant energy consumes oxygen too quickly for diffusion to maintain an adequate level of oxygenation, and dose-response curves obtained are those characteristic of hypoxia. • For bacteria, a dose-rate effect in this region is firmly established and first demonstrated by Dewey and Boag (1959) who found an anoxic-like response of bacteria irradiated in 1 %oxygen at ultra-high dose-rates. This was confirmed by others (e.g. Epp et al., 1968), who found a biphasic (“hockey –stick”) type of response. • Subsequently, this was confirmed by Berry et al. (1969) irradiating mammalian cells equilibrated in air : Both radiation regimes were equally effective at low doses, but for doses in excess of about 5 Gythe pulsed ultrahigh dose-rate X rays were less effective for a given dose than g-rays. Beyond the break-point, the slope of the survival curve for the pulsed radiation, even in the presence of atmospheric air, was less than the slope of the 60Co g-ray survival curve by a factor of about 2·5; i.e. the response is characteristic of anoxia. • The hope was that this would constitute a means of killing cells which would be independent of oxygen tension-i.e. tat sufficiently high dose rates even aerated cells would behave as if they were anoxic, hence he same survival curve wouId apply to aerated or hypoxic cells. This would represent an ideal radiation modality for radiotherapy since the protection afforded to some tumour cells by their hypoxia would be completely eliminated. • However, the total doses required to use up local oxygen are too high, and the instantaneous dose-rates enormous, so that interest in this development for the clinic did not materialize (so far, at least)

  16. Other UHDR effects-1 • Ultra-high dose rate (UHDR) effect must be associated with specific early time physical interactions and subsequently generated radicals. The key issue is not only the spatial proximity but the temporal coincidence of a spur to DNA (simultaneity), which is limited by the spur lifetime (in theorder of ns). For low-LET radiation, a spur coincidence would involve multiple tracks within the short lifetime of a spur, hence an ultra-high dose rate effect attributed to a coincidence of spurs is unlikely. • Forions such as 12C, the single track picture makes simultaneity of spurs more likely so that ultra-high dose rate effects in radiobiology, if they exist, are more likely to occur in response to high-LET irradiation. (Ledingham et al., 2014)

  17. Other UHDR effects-2 • Low-emittance gives rise to sharper penumbra of the treatment fields. • In high-fluenceand ultra-short pulse delivery, the stopping power can be best described by the dielectric response of the target material • In addition to the binary collisions as described by the Bethe-Bloch equation, there are also collective oscillations induced on the target by the impinging ions. • If the ion clusters come in very short pulses or wavetrains, the collective wakes set off by the first cluster affect as a retarding force the trailing one. This retardation by the collective wakes (vicinage effect) result in an increase of the stopping power • Obcemea et al, NIM A (2016) (Malka et al., 2010) Interactions between relativistic electron bunches and water bulk on early radiation damages in nascent ionization clusters are crucial.Thehigh energy radical femtochemistry of water molecules strongly argues for a quantum character pre-eminence of ultrafast radiation damages in native tracks. Considering the time-space relationship of an energy deposition by high-energy ionizing radiation (MeV) in aqueous environment, in less than 1016 s, energy quanta (around 200 and 20 eV) are delivered in nanometric tracks or spurs (white circle). The early behaviourof secondary electrons inside newly formed ionization clusters is dependent on the excess energy relaxation occurring in the range 1014–1012s. In the prethermal regimeradiation damages of biomolecules may occur to a great extent

  18. Radiation-induced premature senescence (PS)

  19. Health implications of PS: • Senescence–Associated Secretory Phenotype (SAPS): secretion of factors released by cells undergoing PS have been associated with either the inhibition or the promotion of cellular proliferation in surrounding tumour cells • Tsai et al., Low-dose radiation-induced senescent stromal fibroblasts render nearby breast cancer cells radioresistant. Radiat Res (2009) • Davaloset al., Senescent cells as a source of inflammatory factors for tumor progression. Cancer Metastasis Rev., 29, 273-283 (2010) • Day et al., Radiation-induced accelerated senescence: A fate worse than death? Cell Cycle, 2014

  20. A low dose of 12C ions with LET values comparable to those experienced by healthy cells at the beam entrance channels causes an early occurrence of PS in HUVEC cells and is much more effective than photons (top). The proliferative potential of control and 2 Gy-irradiated osteosarcoma cells assayed by delayed plating efficiency is reduced by medium from senescing cells (bottom). Such a reduction in clonogenic survival increases with the dose, hence the amount of PS induced in directly exposed HUVEC cells.

  21. Radiation-induced cardiovascular disease Lowe et al., Epigenetic clock analyses of cellular senescence and ageing Oncotarget, 2016

  22. LULI (Laboratoire pour l'Utilisation des Lasers Intenses)

  23. Datasets • Laser-driven proton beam, DR=~109Gy/s (LULI) • Pulsed therapeutic proton beam, DR=2Gy/s(INFN-LNS)  obtained by means of a chopper (capacitor) able to produce a 50ms beam pulse with 500mHz frequency. • Therapeutic proton beam, DR=0,03Gy/s(INFN-LNS) • X-rays, 250 kVp(University of Naples)

  24. Experimental setup-1 • A 100mm long permanent magnet with a constant magnetic field (~0.9T) on a 50x4mm gap angularly dispersed the multi-MeV proton beam produced in the laser-target interaction. • Protons through a Kapton window were then transported onto the biological sample. • An ad hoc pipe re-entrance ensured appropriate dose values to be reached.

  25. Experimental setup-2 • Biological sample seeded in three dots of 3x5 mm2 each • This configuration allowed the simultaneous exposure of three samples(at different energies and dose values). • After exposure, the cells from each dot were treated and examined independently.

  26. The customized sample dish was composed of three elements: an inner component and two twin external components (to which 3-mm thin mylarsheets are attached) screwed to the top and bottom of the inner part . • The dish was equipped with an automated syringe system in order to be able to remove the cell media during irradiation and to re-inject it immediately post irradiation.

  27. Overview of the setup

  28. Dosimetry -1 • The LULI dosimetry set-up configuration involved the employment of different particle detectors such as radiochromic films (RCF), image plates (IP) and nuclear track detectors CR-39s. • Prior the cell irradiation, low-sensitivity HD-V2 RCF were positioned inside the target chamber at about 5 cm from the proton source, in order to study the beam input properties, in terms of energy and angular distribution, before it entered the transport and delivery system. • HD-V2 stacks were wrapped in a 12-µm Al foil in order to reduce the electron, heavy ion and photon contributions to the detector’s signal.

  29. RCF stack obtained irradiating a 25-µm Au target. Each layer reports the correspondent proton energy obtained from SRIM calculations. • The energy values written on each RCF layer are obtained using the Stopping Range of Ions in Matter (SRIM) software toolkit

  30. Figure shows the energy distribution as a function of particle abundance correspondent to the laser interaction with the three Au targets thicknesses (10, 25 and 50 µm) employed in this experiment. • The energy spectrum corresponding to the 50-µm Au target is characterized by a lower proton fluence and also shows an energy cut-off unlike the other Au targets . The energy spectrum of the 10 and 25 µm target thicknesses are similar, but the spatial distributions of protons in terms of homogeneity are not.

  31. Comparison between the two 10th layers of the RCF stacks obtained from the laser interaction with the 10 (left) and 25 µm (right) Au targets, which corresponds to the 10 MeV proton contribution. • In both cases, a higher fluenceoccurred at one edge (due to the intrinsic angular distribution of particles with different energies), which decreases towards the periphery more smoothly for the 25 µm target. Therefore, in the experimental campaign we used the 25 and 50 µm Au targets to be able to irradiate the samples with different dose values while the 10 µm Au target was dismissed due to the poor beam homogeneity it gave rise to.

  32. Left: detectors positioning in the irradiation configuration. Right: scheme of the detectors sequence behind the biological sample. • To evaluate the beam dose output values, high-sensitivity EBT3 RCFs were used. They were placed outside the target chamber, just behind the biological sample, in an ad hoc frame, as to provide a shot-to-shot characterization of the irradiation dose. • Image plates (IPs) were employed for the beam energy evaluation. CR-39 nuclear track detectors, instead, were used to estimate the particles fluence and homogeneity, and to cross-calibrate the IPs. During sample irradiation, IPs or CR39s were placed behind the EBT3 RCFs, which were in turn positioned behind the biological sample. • This allowed to obtain a dose evaluation (by means of the EBT3) as well as an energy distribution along the beam dispersion axis (by means of EBT3, IP/CR39 and Al filters) simultaneously with the irradiation of the biological sample irradiation. This way it was possible to attribute each dose to each position, hence the cell dots • An Al filter was placed between the RCF and the IP/CR-39. Several Al filters were employed in order to stop particles of different energies according to their thickness , and to be able to obtain energy cut-off positions on the beam dispersion axis (y) from the IP/CR-39 output analysis.

  33. Energy values corresponding to the cell dots positioning in relation to the two different entrance slit apertures.

  34. The laser shots sequence of the LULI experimental campaign. Mean dose values and related standard deviations, measured on a 3x5 mm2 area, in correspondence to the targets’ thicknesses, the radiobiological end-points and the slit entrance apertures are also shown • No significant variation was observed in the overall dose values obtained employing the 310 and 510 µm slit entrance apertures. This is probably due to the beam fluctuation and/or target inhomogeneity. A dose-rate of about 3×109 Gy/s was estimated.

  35. The energy differences within the dot-containing dish region did not correspond to a LET variation able to elicit significantly different biological effects (at least as far as traditional radiobiology is concerned). Hence, laser-driven beam data were pooled and evaluated as a function of dose. • In the pulsed and conventional proton beam experiments, irradiations were carried out using beams having as LET the mean value of the laser-driven irradiation LETs. • The energy of the LNS-INFN proton beam employed for the pulsed and conventional irradiations was attenuated by means of degraders in order to obtain LET values comparable to the laser-driven ones.

  36. Endpoints What does cellular survival mean? Every irradiated cell is considered survived if keeps intact its reproductive capacity, developing a colony  Puck and Marcus in the ‘50s give an operative definition of colony  agglomeration of at least fifty cells, corresponding to 5-6 cells division post irradiation. Radiobiologicaltests: Cellular survival Clonogenicassay Cellular senescence  β-galactosidase assay Gold standard assay for studying cellular radio-sensitivity, basis for radiation therapy treatment planning & reference test for other radiobiological assays. What doescellular senescence mean? Radiation induced senescence is called: “Stress-Induced Premature Senescence” (SIPS)  Cells are metabolically active but they irreversibly lose proliferative capacity. Accumulation of senescent cells, may have consequence in vivo since they can affect normal tissue (and organ) integrity and/or function as well as increase the risk of secondary cancer.

  37. Cell survival • Within the errors there is no statistically significance between the three proton-derived cell survival dose-response curves • All three proton irradiation conditions are, however, slightly more effective than X-rays at causing cell death. • Cellular survival analysis shows that the extremely high dose-rates and pulsed nature of laser-driven beams may at least be considered as effective as the conventionally accelerated protons 

  38. Radiation-induced premature senescence-1

  39. Radiation-induced PS-2

  40. Radiation-induced PS-3 • Senescence induced by the laser-driven and the pulsed beam has a similar trend. • Senescence induced by conventional and X-ray beam has a similar trend.

  41. Conclusions • Cellular survival: • Extremely high dose-rate pulsed laser-driven proton beams appear to exhibit a similar RBE to therapeutic protons in terms of cell death induction • Cellular senescence was assessed for the first time after laser acceleration and compared to therapeutic proton beams: • Was induced less effectivelyat low doses • Was induced more effectively at typical therapeutic doses • Laser-driven proton beams are less effective than therapeutic protons at causing PS at doses relevant to the healthy tissue while being more effective at (almost) therapeutic doses: if confirmed this of significance in radiotherapy scenarios. 

  42. Our results in context • The overall majority of in vitro studies for different cell-lines and making use of different sources confirm that in the therapeutically relevant dose range of a few Gy, even if applied in a single pulse of only few nanoseconds duration, non-linear radiobiological effects due to simultaneous multiple damages in cells and, thus, below any timescale of repair mechanisms are unlikely to arise. Only two exceptions: • Achayra et al.,Radiat Environ Biophys (2011): decrease in MN formation after single pulse of electronsbut not after multiple pulses (106 to 108 Gy/s) • Schmid et al, RadiatRes (2011): slight decrease in effectiveness at causing (some types of) chromosome aberrationsafter nanopulsed protons (conventionally accelerated) Experiments in vivo (still too few): no effect on tumour growth delay • Bruchneret al (2014) RadiatOncol 2014, (electrons) • Oppelt et al Radiat Environ Biophys 2015 (electrons) • Zlobinskaya, Radiat Res, 2016 (protons) • However, Favaoudon et al , Sci Transl Med (2014) report in vivo sparing of normal tissue in mice albeit with «only» 40Gy/s electrons (not pulsed), but Laschinsky et al, RadiatEnviron Biophys (2016) found no effectin vitro on normal humancellswithhighlypulsedelectrons

  43. What mechanisms at play for PS following ultra-high dose rate and ultra-short pulsed irradiation?

  44. Thanks to: • Dr. Francesca M. Perozziello • University of Naples Federico II & • INFN Naples Section • INFN-LNS staff, Catania • Queen’s University staff, Belfast • EcolePolytechnique staff, Paris

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