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International audience; Background: Resistance to radiotherapy is frequently encountered in patients with glioblastoma multiforme. It is caused at least partially by the high glutathione content in the tumour tissue. Therefore, the administration of the glutathione synthesis inhibitor Buthionine-SR-Sulfoximine (BSO) should increase survival time.Methods: BSO was tested in combination with an experimental synchrotron-based treatment, microbeam radiation therapy (MRT), characterized by spatially and periodically alternating microscopic dose distribution. One hundred thousand F98 glioma cells were injected into the right cerebral hemisphere of adult male Fischer rats to generate an orthotopic small animal model of a highly malignant brain tumour in a very advanced stage. Therapy was scheduled for day 13 after tumour cell implantation. At this time, 12.5% of the animals had already died from their disease.The surviving 24 tumour-bearing animals were randomly distributed in three experimental groups: subjected to MRT alone (Group A), to MRT plus BSO (Group B) and tumour-bearing untreated controls (Group C). Thus, half of the irradiated animals received an injection of 100 mu M BSO into the tumour two hours before radiotherapy. Additional tumour-free animals, mirroring the treatment of the tumour-bearing animals, were included in the experiment. MRT was administered in bi-directional mode with arrays of quasi-parallel beams crossing at the tumour location. The width of the microbeams was approximate to 28 mu m with a center-to-center distance of approximate to 400 mu m, a peak dose of 350 Gy, and a valley dose of 9 Gy in the normal tissue and 18 Gy at the tumour location; thus, the peak to valley dose ratio (PVDR) was 31.Results: After tumour-cell implantation, otherwise untreated rats had a mean survival time of 15 days. Twenty days after implantation, 62.5% of the animals receiving MRT alone (group A) and 75% of the rats given MRT + BSO (group B) were still alive. Thirty days after implantation, survival was 12.5% in Group A and 62.5% in Group B. There were no survivors on or beyond day 35 in Group A, but 25% were still alive in Group B. Thus, rats which underwent MRT with adjuvant BSO injection experienced the largest survival gain.Conclusions: In this pilot project using an orthotopic small animal model of advanced malignant brain tumour, the injection of the glutathione inhibitor BSO with MRT significantly increased mean survival time.

International audience; Synchrotron-generated microplanar beams (microbeams) provide the most stereo-selective irradiation modality known today. This novel irradiation modality has been shown to control seizures originating from eloquent cortex causing no neurological deficit in experimental animals. To test the hypothesis that application of microbeams in the hippocampus, the most common source of refractory seizures, is safe and does not induce severe side effects, we used microbeams to induce transections to the hippocampus of healthy rats. An array of parallel microbeams carrying an incident dose of 600 Gy was delivered to the rat hippocampus. Immunohistochemistry of phosphorylated gamma-H2AX showed cell death along the microbeam irradiation paths in rats 48 hours after irradiation. No evident behavioral or neurological deficits were observed during the 3-month period of observation. MR imaging showed no signs of radio-induced edema or radionecrosis 3 months after irradiation. Histological analysis showed a very well preserved hippocampal cytoarchitecture and confirmed the presence of clear-cut microscopic transections across the hippocampus. These data support the use of synchrotron-generated microbeams as a novel tool to slice the hippocampus of living rats in a minimally invasive way, providing (i) a novel experimental model to study hippocampal function and (ii) a new treatment tool for patients affected by refractory epilepsy induced by mesial temporal sclerosis.

Cisplatin (CDDP) is an anticancer drug widely used in clinic for the treatment of several solid tumours, despite its high effectiveness treatment is still limited by severe side effects and by inherited or acquired resistance phenomena. These drawbacks have stimulated the search of alternative strategies based on different metals offering a better toxicity profile while maintaining the same level of efficacy as platinum-based treatments. [1, 2] Recently we evaluated the cytotoxicity and neurotoxicity of our newly developed promising water soluble anticancer complexes ([Cu(PTA)4]PF6, [Cu(thp)4]PF6, [Au(PTA)4]PF6, [Au(thp)4]PF6) using CDDP as reference drug. The cytotoxicity was evaluated.on A549 non-small cell lung cancer (NSCLC) and IGROV-1 ovarian human cancer cells while the neurotoxicity was tested on dorsal root ganglia organotypic cultures. In our model CDDP resulted neurotoxic at concentrations achievable in plasma of patients treated with the same drugs. Similarly the gold-based compound [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 calculated for the tested cancer cell lines. On the contrary, both copper-based compounds and [Au(thp)4]PF6 were neurotoxic at higher concentrations with respect to the IC50 obtained in tumour cell lines tested. We then tested at the ID17 beamline of the ESRF the efficacy of synchrotron radiation (SR) to trigger the Auger effect in IGROV-1 cells containing a high Z-number element. Irradiation of cells pre-treated with CDDP or [Cu(PTA)4]PF6 concentrations allowing roughly 90% of cell survival induced an enhancement in cellular death with respect to drug and irradiation alone. With the other compounds no cell death enhancement was observed. Our results suggest that SR-enhanced CDDP activity might allow the use of a reduced dose of CDDP thus achieving side effects minimization due to the exposure of normal cells/tissues to less toxic doses. Furthermore, considering the anticancer activity and the neurotoxic profile of [Cu(PTA)4]PF6, our data suggest that copper-based drugs represent new and promising compounds in anticancer treatment also in combination with SR. Acknowledgments Authors thanks the COST action TD1205 References: [1] Ceresa, C.; Bravin, A.; Cavaletti, G.; Pellei, M. and Santini C. Curr. Med. Chem. 2014, 21, 2237-2265. doi: 10.2174/0929867321666140216125721 [2] Ceresa, C; Nicolini, G; Semperboni, S; requardt, H; Le Duc, G; Santini, C; Pellei, M; Bentivegna, A; Dalprà, L; Cavaletti, G; Bravin, A. Anticancer Res. 2014, in press.

Cisplatin is one of the most effective metal-based anticancer agents, targeting a large number of solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. In this work we investigated the in vitro activity and neurotoxicity of three anticancer complexes: [Cu(PTA)4]PF6, [Cu(thp)4]PF6 and [Au(PTA)4]PF6. Neurotoxicity was evaluated by embryonic rat dorsal root ganglia (DRG) organotypic culture model. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by fluorimetric assay. After 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug, we evaluated their ability to affect the proteasome machinery in DRG neurons. The copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4] PF6, induces a significant inhibition of proteasome activity even at concentration lower than the IC50. Furthermore, based on the content in heavy metal-based atoms, the complexes used in this study are suitable candidates for Photon Activation Therapy (PAT). Pretreatment of IGROV-1 cells with [Cu(PTA)4]PF6 induces an increase cell death with respect to drug or synchrotron (SR) alone. Furthermore the SR/[Cu(PTA)4]PF6 combinational treatment induced an increase in DNA damage with respect to single treatments. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

Background. Glioblastoma multiforme (GBM) is one of the deadliest cancers since it shows a very limited sensitivity to chemo- and/or radiotherapy. Experimental evidence suggests that the presence in the tumour environment of GBM stem-like cells might be particularly relevant to confer this extraordinary resistance to treatment. Aim. In this study, we aimed at providing a proof-of-concept of the efficacy of Photo Activation Therapy (PAT) using monochromatic X-rays, issued from a synchrotron radiation (SR) source, in killing human glioma stem cells pre-treated with cisplatin. We have also investigated if lethality of this treatment was superior to that achievable with conventional X-ray irradiation. Material and Methods. Cisplatin was chosen as the carrier of platinum atoms in the cells based on previous experience in glioma and non-glioma cancer cells; the irradiation was performed by delivering 1-8 Grays using beams of energies just above or below the platinum K-shell edge (78.39 keV) or with a conventional X-ray source. Cells were exposed to low drug concentrations allowing 90% or more of GBM stem-like cells survival thus mimicking the unfavourable tissue distribution generally achieved in GMB patients. Results. Under these experimental conditions a significant enhancement in cell lethality was observed using SR with respect to conventional X-ray irradiation. Conclusions. Our results provide additional support to the suggestion that PAT deserves to be further explored in relevant in vivo models based on GBM stem-like cells.

Glioblastoma multiforme (GBM) is one of the deadliest cancers characterized by very limited sensitivity to chemo- and/or radiotherapy. The presence of GBM stem-like cells in the tumor might be relevant for GBM treatment resistance.To provide a proof-of-concept of the efficacy of photon activation therapy (PAT) using monochromatic synchrotron radiation (SR), in killing GBM stem cells pre-treated with cisplatin.Irradiation was performed using a 1-8 Gy dose range and energies just above or below the platinum K-shell edge (78.39 keV) or with a conventional X-ray source. Cells were exposed to drug concentrations allowing 90% cell survival, mimicking the unfavourable tissue distribution generally achieved in GMB patients.a significant enhancement in cell lethality was observed using SR compared to conventional X-ray irradiation.PAT deserved to be further explored in in vivo models based on GBM stem-like cells.

Background. Cisplatin is one of the most efficient metal-based anticancer agents, targeting several solid tumours. Despite its efficacy, cisplatin treatment is still limited by severe side effects such as neuro-, hepato- and nephro-toxicity and by resistance phenomena, only partially overcome by the use of new platinumdrugs (i.e. oxaliplatin and carboplatin). These problems have stimulated the research and development of alternative therapeutic strategies based on different heavy metals. Furthermore, heavy metal-based anticancer complexes represent suitable candidates to be used in Photon Activation Therapy (PAT). Aim. We investigated the in vitro activity and neurotoxicity of three anticancer complexes: [Cu(PTA)4]PF6, [Cu(thp)4]PF6 and [Au(PTA)4]PF6. Furthermore we investigated the efficacy of synchrotron radiation (SR) to trigger the Auger effect in human IGROV-1 ovarian cancer cells pre-treated with [Cu(PTA)4]PF6. Material and Methods. Neurotoxicity was evaluated by embryonic rat dorsal root ganglia (DRG) organotypic culture model. Furthermore the extent of proteasome inhibition in rat embryonic DRG neurons was evaluated by fluorimetric assay. The efficacy of SR in combination with [Cu(PTA)4]PF6 was evaluated, in terms of cell survival, by standard clonogenic assay. DNA damage induced by the combinational treatment was also tested by RAD51 nucelar foci counting and BRCA1 nuclear expression. Results. After 48 hours of treatment, both copper-based compounds were not neurotoxic even at higher concentrations with respect to the IC50 obtained in A549 and IGROV-1 human cancer cells while [Au(PTA)4]PF6 was neurotoxic at lower concentration than IC50 in cancer cell lines tested. Since the ubiquitin-proteasome system has been identified as molecular target in cancer cells for the heavy metal based-drug, we evaluated their ability to hinder the proteasome machinery in DRG neurons. Both copper-based compounds, that are not neurotoxic, do not inhibit proteasome activity in DRG neurons. On the contrary, the neurotoxic complex [Au(PTA)4]PF6, induces a significant inhibition of proteasome activity even at concentration lower than the IC50. PAT experiment in IGROV-1 cells showed that the pre-treatment with [Cu(PTA)4]PF6 induces an increase in cell death with respect to drug or SR alone. Furthermore the SR/[Cu(PTA)4]PF6 combinational treatment induced an increase in DNA damage with respect to single treatments. Conclusions. Our results, together with the low IC50 of the copper compounds compared to the one observed for cisplatin, suggest them as promising compounds in anticancer treatment. Finally our results suggest the potential use of [Cu(PTA)4]PF6 in PAT providing support to further studies in reliable in vivo animal models.

Cisplatin (CDDP) is an anticancer drug widely used in clinic for the treatment of several solid tumours. Despite its high effectiveness CDDP treatment is still limited by severe side effects and by inherited or acquired resistance phenomena, only partially overcome by the use of new platinum drugs (i.e. oxaliplatin and carboplatin). These drawbacks have stimulated the search of alternative strategies based on different metals offering a better toxicity profile while maintaining the same level of efficacy as platinum-based treatments. In this study we evaluated the cytotoxicity and neurotoxicity of new promising water soluble anticancer complexes based on copper [Cu(PTA)4PF6] [Cu(thp)4][PF6] and gold [Au(PTA)4PF6] [Au(thp)4][PF6] on A549 non-small cell lung cancer (NSCLC) and IGROV-1 ovarian human cancer cells. IC50 at 48 hours of treatment was calculated by SRB assay while the neurotoxicity was tested evaluating the interference of the compounds with neurite elongation. CDDP was used as drug reference with respect to our newly developed drugs. In our model CDDP resulted neurotoxic at concentrations achievable in plasma of patients treated with the same drugs. Similarly the gold-based compound [Au(PTA)4PF6] was neurotoxic at lower concentration than IC50 calculated for the tested cancer cell lines. On the contrary, both copper-based compounds and [Au(thp)4][PF6] were neurotoxic at higher concentrations with respect to the IC50 obtained in tumour cell lines tested. We then tested the efficacy of synchrotron radiation (SR) to trigger the Auger effect in A549 and IGROV-1 cells containing a high Z-number element, like platinum, gold, and copper. CDDP and the new metal-based drugs were used as carrier of heavy Z-elements. The irradiation was performed with monochromatic beams above the K-shell edge of the different elements. Using keV X-rays, the high photoelectric cross sections of the heavy-Z elements result in substantial interactions, producing fluorescence light and free electrons. The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, where a high-fluence monochromatic beam adjustable from 20 to 180 keV is available. Irradiation of cells pre-treated with CDDP or [Cu(PTA)4PF6] concentrations allowing roughly 90% of cell survival induced an enhancement in cellular death with respect to drug and irradiation alone. With the other compounds no cell death enhancement was observed. Finally we studied the homologous recombination repair pathways. SR in combination with CDDP resulted in a significative nuclear relocalization of RAD51 and BRCA1 proteins with respect to untreated control A549 and IGROV-1 cells as well as in cells treated with SR or CDDP alone. Similar results were observed in IGROV-1 cells pre-treated with [Cu(PTA)4PF6]. Our results suggest that SR-enhanced CDDP activity might allow the use of a reduced dose of CDDP thus achieving side effects minimization due to the exposure of normal cells/tissues to less toxic doses. Furthermore, considering the anticancer activity and the neurotoxic profile of [Cu(PTA)4PF6], our data suggest that copper-based drugs represent new and promising compounds in anticancer treatment also in combination with SR.

International audience; Owing to their large range of properties which can be accurately tuned by the chemical composition, the shape and the dimensions, multifunctional nanoparticles appear as promising candidates for imageguided therapy. For achieving this goal, we developed the synthesis of gold nanoparticles which are designed for combining multimodal imaging (magnetic resonance imaging (MRI), scintigraphy (SPECT), ultrasound imaging (echography) and X-ray imaging) and radiotherapy [1-5]. Ultrasmall gold nanoparticles were synthesized by reducing gold salt in presence of various highly hydrophilic dithiolated polyaminocarboxylate ligands (linear (DTDTPA) and macrocyclic (TADOTA, TADOTAGA) ligands). They are composed of a gold core (mean diameter of ~ 2.5 nm) encapsulated within an organic shell of ligands (DTDTPA, TADOTA or TADOTAGA). The gold core is expected to provide a strong X-ray absorption whereas the ligands of the organic shell were chosen for their propensity to entrap gadolinium ions (for MRI) and radionuclides (for SPECT). Since the passive accumulation of the gold nanoparticles in the tumor depends on the nature of theligands, the possibility to follow up the gadolinium chelate-coated gold nanoparticles by MRI is therefore very useful for determining the most opportune delay between intravenous injection and irradiation. Owing to the radiosensitizing effect of the gold core, the combination of microbeamradiation therapy (MRT) and gold nanoparticles based MRI contrast agents led to a great increase in lifespan of the 9L gliosarcoma-bearing rats in comparison to non-treated animals and animals treated only by MRT [5]. Finally, this study demonstrated that gadolinium chelate-coated gold nanoparticles exhibit a real potential for image-guided microbeam radiation therapy.

Cisplatin is an antineoplastic drug widely used for the treatment of several solid tumours. However, the side effects related to cisplatin-based anticancer therapy often outweigh the benefits. Therefore, the identification of new anticancer strategies able to offer a better toxicity profile while maintaining the same level of efficacy as platinum-based treatments would be highly desirable. We assessed the efficacy of synchrotron radiation in triggering the Auger effect in human A549 non-small cell lung cancer and IGROV-1 ovarian cancer cells pre-treated with cisplatin. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). On cisplatin-treated cells, at concentrations allowing 80 percent of cell survival with respect to controls, no differences were observed in cell viability when they were irradiated either above or below the K-shell edge of platinum, suggesting that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation. At lower cisplatin concentrations allowing 95-90 percent of cell survival, an enhancement in cellular death with respect to conventional irradiation conditions was clearly observed in all cancer types when cells were irradiated with beams either above or below the platinum K-shell edge. Our results lend additional support to the suggestion that the Photon Activation Therapy in combination with cisplatin treatment should be further explored in relevant in vivo models of glioma and non-glioma cancer models.

International audience; Ultrasmall gadolinium-based nanoparticles (GBN, hydrodynamic diameter < 5 nm) were recently developped. Since they are designed for combining renal clearance, magnetic resonance imaging (MRI) and radiosensitization, these nanoparticles appear as attractive agents for image-guidedradiotherapy. Their efficiency to improve the survival time of tumor bearing animals rests on the possibility to determine, from the data collected by MRI, the delay between the irradiation and the intravenous injection of GBN. The attempts to determine this delay were assessed by combining MRT experiments in parallel with MRI and it provided unexpected results. In this talk, we will summarize these results that were achieved through the long term project MD606 (ANR Theraguima) at the ESRF including survival curves of animals as a function of (i) the time delay between injection and irradiation, (ii) the combination between nanoparticles and temozolomide and (iii) the comparison with the radiosensitization issued by the use of Gd chelates instead of GBN. Additionally, the biodistribution of the GBN within the tumor will be presented through MRI results and XRF results (ID21). On going experiments are now focused on the comparison between the efficiency of the GBN using MRT and in parallel a 6MeV irradiator at the local hospital.

Cisplatin is an antineoplastic drug widely used in clinic for the treatment of several solid tumours. However, at times, the side effects related to cisplatin-based anticancer therapy often outweigh the benefits. Several of these side effects are now manageable with effective treatment planning and supportive care strategies (e.g. anti-emetics, hematopoietics growth factors, physical treatments, hyperhydration), but others are still unsolved, can be dose-limiting and represent an unmet clinical need, for instance nervous system damage. Therefore, the identification of new anticancer strategies able to offer a better toxicity profile maintaining the same level of efficacy as platinum-based treatments would be highly desirable. We tested the efficacy of synchrotron radiation to trigger the Auger effect in the living human A549 non-small cell lung cancer (NSCLC) and IGROV-1 ovarian cancer cells pre-treated with cisplatin. Furthermore we tested the anticancer treatment in human VIPI glioblastoma stem cells that seem to be one of the most important determinants in the extraordinary resistance of GBL to any treatment tested so far. The experiments were carried out at the ID17 beamline of the European Synchrotron Radiation Facility, where a high-fluence monochromatic beam adjustable from 20 to 80 keV is available. Cisplatin was chosen as the carrier of platinum atoms in the cells because of its alkylating-like activity and the irradiation was done with monochromatic beams above and below the platinum K-shell edge (78.39 keV). Control cell survival curves were comparable with those obtained for the same cells under conventional irradiation conditions. On cisplatin-treated cells at a concentrations inducing 80% of cell survival with respect to the control, no differences were observed in cell survival when cells were irradiated either above or below the K-shell edge of platinum, suggesting that cisplatin toxicity can mask the enhancement of cell death induced by the irradiation above the K-shell edge. At lower cisplatin concentrations, determining 95-90% of cell survival an enhancement in the cellular death was observed, with respect to conventional irradiation conditions was observed when cells were irradiated with beams either above or below the platinum K-shell edge. Our results suggest that SR-enhanced CDDP activity might allow the use of a reduced dose of CDDP thus achieving side effects minimization due to the exposure of normal cells/tissues to less toxic doses. Furthermore SR-enhanced CDDP activity might enhance CDDP effect on cancer stem cells obtained from human glioblastoma. Reliable in vivo animal models might be investigated in future experiments to assess activity and toxicity of the proposed treatment in cancer-bearing mice.