Enhancement of survival of 9L gliosarcoma bearing rats following intracerebral delivery of drugs in combination with microbeam radiation therapy

https://doi.org/10.1016/j.ejrad.2008.04.049Get rights and content

Abstract

Microbeam radiation therapy (MRT) is a form of radiosurgery first dedicated to the treatment of brain tumors. It uses arrays of synchrotron generated X-rays microbeams of very high doses (typically 625 Gy). Microbeams are typically few micrometers large (25 μm) and few hundred micrometers spaced (200 μm). Previous experiments have shown that despite a good tumor eradication rate (5/11), a 100-μm spacing unidirectional irradiation (skin dose 625 Gy, width 25 μm) was too invasive for normal tissue. On the contrary, a 200-μm spacing unidirectional irradiation preserved healthy tissue with a low tumor eradication rate (2/32). The purpose of this study was to enhance the potential of the 200 μm spacing irradiation protocol. After diagnosis of the tumor by MRI, 9L tumor-bearing rats were laterally irradiated with 51 microbeams (625 Gy, 25 μm, 200 μm) 14 days after implantation. Three drugs (Gd-DTPA, CisPt, temozolomide) were tested, after intratumoral injection at the theoretical center of the tumor. Control rats displayed a median survival time of 19 days. There was no significant difference between drug-treated rats and control group. Irradiated animals showed an increase in life span (ILS) of 60.5%. Interestingly, the ILS increased to 131.6% and 1/6 rat survived more than 1 year in case of MRT combined with gadolinium injection. These results showed that the synergy between gadolinium injection (acting as a dose enhancer) and MRT improved significantly the life span of tumor bearing rats (more than a factor 2).

Introduction

Gliomas are among the most frequent primary brain tumors in adults, with an incidence of approximately 5–11/100,000 among the general population per year in industrial countries. Despite a combination of surgery, chemotherapy and radiotherapy, the treatment of high-grade gliomas remains palliative and the median survival for patients with glioblastoma multiforme is less than 1 year. Synchrotron facilities, in particular the European Synchrotron Radiation Facility (ESRF) emerged as complementary tools for research in radiotherapy. Especially, microbeam radiation therapy (MRT) differs from other techniques by employing smaller beams (typically few microns wide) than the smallest radiation beam used in hospitals (typically several millimeters). This concept is technically feasible thanks to the use of highly intense synchrotron X-ray beams, with a sufficient high energy (allowing a deep penetration in tissue and a high skin entry dose typically equal to 600 Gy), a negligible divergence (allowing the production of sharply defined beam edges in tissues) and high flux (allowing a fast irradiation process in order to prevent motion artifacts of the subject). Previous experiments performed on the brain of adult rats [1], suckling rats [2], duck embryos [3], and piglets [4] highlighted a sparing effect on normal tissues when using microbeams. In parallel, it was shown that MRT protocols can preferentially ablate 9L brain tumors [5], [6], EMT-6 carcinoma [7] and SCCVII carcinoma [8].

In a recent work [9], we obtained results in agreement with other studies performed at the ESRF [10] when irradiating 9L glioma in unidirectional mode (skin entry dose: 625 Gy, beam width: 25 μm, spacing: 200 μm). Moreover, we demonstrated that the balance between the sparing of normal tissue and the curing of the tumor was depending on the spacing between the microbeams (625 Gy skin entrance, 25 μm width of microbeams). Although the curing potential of the 100 μm was shown to be high, it provokes significant damages to normal tissues. Contrarily, despite a lower tumor control by using the 200-μm protocol, the sparing effect of normal tissues for this protocol allows more possibilities of improvements.

Consequently, to determine whether the potential of these irradiation parameters could be enhanced while keeping the sparing of surrounding tissue, we tested the intratumoral injection of drugs in combination with the MRT treatment. Intratumoral injection allows: (i) to overpass the systemic toxicity that might be due to the drug injection, (ii) to bypass the blood–brain and blood–tumor barrier and (iii) to increase the local concentration of the drug [11]. It was proven to be successful even when manually injected 1 day before treatment by stereotactic synchrotron radiation beam (SSRT) [12] on the F98 glioma model.

Temozolomide [13] has proven efficacy against malignant brain tumor and admitted as a standard for the treatment of glioblastoma when concomitant with radiotherapy. Especially, the median survival time of patients was elongated from 12.1 months to 14.6 months with a survival rate at 2 years equal to 26% against 8% for radiotherapy only. Oral administration is quite difficult in the case of animals while intratumoral injection was proven to be feasible [14]. On the contrary, dose enhancement effect is a pure physical process. Drugs containing high Z elements can be selectively accumulated in tumor prior to any irradiation protocol. The increase of the local deposited dose goes in hand with an increase of the radiobiological injury, in direction of the tumor cells. In the case of the polychromatic beam use for the MRT at the ESRF [15], one of the most suitable element would be gadolinium (Z = 64). Another drug, CisPt, already widely use in clinical routine, has been highlighted recently as a highly interesting drug, creating both a cytotoxic and dose enhancement effect in parallel, by using a monochromatic synchrotron source [12].

The purpose of this work was to screen any efficacy of the MRT technique when combined with intratumoral drug delivery. Temozolomide, Gd-DTPA, and CisPt were chosen for creating a cytotoxic effect, a dose enhancement effect, and both, respectively. Experiments were performed 14 days after implantation of 9L gliosarcoma cells in rats. All series were performed during the same experimental sessions. The efficiency of the combination between drugs and MRT was assessed by a fine correlation between survival curves, follow-up of animals and histological analysis.

Section snippets

Intracerebral model in rat brain

The 9L gliosarcoma cells were grown with Dulbecco's modified Eagle medium (DMEM) (Gibco-Invitrogen-France, Cergy-Pontoise, France) without sodium pyruvate (with 4500 mg/l of glucose and pyridoxine HCl) supplemented with 10% fetal calf serum, 1% penicillin/streptomycin and incubated at 37 °C in a mixture of air–CO2 (95–5%). Animals were anesthetized with 4% isoflurane inhalation followed by an intraperitoneal injection of 400 mg/kg of chloral hydrate.

The male Fisher 344 rats (180–280 g, Charles

Results (Table 1 and Fig. 1)

As shown Fig. 1, the irradiated animals had a MeST of 30.5 days and a MST of 30.6 ± 0.53 days, which corresponds to an increase in life span of 60.5 and 63%, respectively, if compared to untreated controls of the present study (MeST for untreated rats = 19 days). No increase in life span were observed after chemotherapy only (MeST after CisPt injection = 20 days, MeSt after temozolomide injection = 19 days) or after intratumoral injection of Gd-DTPA (MeST after intratumoral injection of GdDTPA = 19

Validation of the 9L model

The MeST of CTRL rats of 19 days is in agreement with similar protocols [5], [6] and with ongoing studies in our lab (MeST = 19 days; mean = 19.7 ± 0.27 days, n = 54). Histological features were consistent with the classification of the 9L tumor as a gliosarcoma [18].

CisPt

No enhancement of the survival curves was observed after CisPt injection. Altogether, on going studies performed in our lab reinforced these observations with a total of 10 rats exhibiting a MeST equal to 21 days when treated with CisPt.

Conclusion

The number of animals contained in each series was chosen to be small since the purpose of the study was to screen few drugs at the same time, on the same tumor model and with the same irradiation conditions and more studies should reinforced these preliminary results. However, the survival curves highlight the potential interest of gadolinium and temozolomide in the context of microbeam radiation therapy against glioma. In the case of gadolinium especially, optimization of the protocols are

Conflict of interest

All authors declare that they have no conflicts of interest.

Acknowledgements

We thank A. Kusak, C. Gommet, C. Clair, E. Siegbahn, G. Roth, E. Bartolami, D. Dallery, C Nemoz, H Requardt and T Brochard for their technical help at any step of the experiment. We wish to address special thanks to Jan Olaf Gebbers for pharmaceutical assistance.

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1

Present address: Turku University Central Hospital, Department of Oncology and Radiotherapy, POB 52, FIN-20521, Turku, Finland.

2

Present address: Centre de Primatologie, ULP Strasbourg, Fort Foch F-67207 Niederhausbergen, France.

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