Proton minibeam radiation therapy (pMBRT) is a novel strategy for minimizing normal tissue damage resulting from radiotherapy treatments. This strategy partners the inherent advantages of protons for radiotherapy with the gain in normal tissue preservation observed upon irradiation with narrow, spatially fractionated beams. In this study, whole brains (excluding the olfactory bulb) of Fischer 344 rats (n = 16) were irradiated at the Orsay Proton Therapy Center. Half of the animals received standard proton irradiation, while the other half were irradiated with pMBRT at the same average dose (25 Gy in one fraction). The animals were followed-up for 6 months. A magnetic resonance imaging (MRI) study using a 7-T small-animal MRI scanner was performed along with a histological analysis. Rats treated with conventional proton irradiation exhibited severe moist desquamation, permanent epilation and substantial brain damage. In contrast, rats in the pMBRT group exhibited no skin damage, reversible epilation and significantly reduced brain damage; some brain damage was observed in only one out of the eight irradiated rats. These results demonstrate that pMBRT leads to an increase in normal tissue resistance. This net gain in normal tissue sparing can lead to the efficient treatment of very radio-resistant tumours, which are currently mostly treated palliatively.

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Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children. Despite intensive research in recent decades the prognosis for patients with metastatic or relapsed diseases has hardly improved. New therapeutic concepts in anti-tumor therapy aim to modulate the patient’s immune system to increase its aggressiveness or targeted effects toward tumor cells. Besides surgery, radiotherapy and chemotherapy, immune activation by direct application of cytokines, antibodies or adoptive cell therapy are promising approaches. In the last years, adoptive transfer of natural killer (NK) cells came into the focus of translational medicine, because of their high cytotoxic potential against transformed malignant cells. A main challenge of NK cell therapy is that it requires a high amount of functional NK cells. Therefore, ex vivo NK cell expansion protocols are currently being developed. Many culturing strategies are based on the addition of feeder or accessory cells, which need to be removed prior to the clinical application of the final NK cell product. In this study, we addressed feeder cell-free expansion methods using common γ-chain cytokines, especially IL-15 and IL-21. Our results demonstrated high potential of IL-15 for NK cell expansion, while IL-21 triggered NK cell maturation and functionality. Hence, we established a two-phase expansion protocol with IL-15 to induce an early NK cell expansion, followed by short exposure to IL-21 that boosted the cytotoxic activity of NK cells against RMS cells. Further functional analyses revealed enhanced degranulation and secretion of pro-inflammatory cytokines such as interferon-γ and tumor necrosis factor-α. In a proof of concept in vivo study, we also observed a therapeutic effect of adoptively transferred IL-15 expanded and IL-21 boosted NK cells in combination with image guided high precision radiation therapy using a luciferase-transduced RMS xenograft model. In summary, this two-phased feeder cell-free ex vivo culturing protocol combined efficient expansion and high cytolytic functionality of NK cells for treatment of radiation-resistant RMS.

This review discusses available clinical and experimental data and the underlying mechanisms involved in trimodality treatment consisting of hyperthermia, cisplatin and radiotherapy. The results of phase I/II clinical trials show that trimodality treatment is effective and feasible in various cancer types and sites with tolerable toxicity. Based on these results, phase III trials have been launched to investigate whether significant differences in treatment outcome exist between trimodality and standard treatment. In view of the clinical interest, it is surprising to find so few preclinical studies on trimodality treatment. Although little information is available on the doses of the modalities and the treatment sequence resulting in the largest degree of synergistic interaction, the results from in vivo and in vitro preclinical studies support the use of trimodality treatment for cancer patients. Animal studies show an improvement in treatment outcome after trimodality treatment compared with mono- and bimodality treatment. Studies in different human tumour cell lines show that a synergistic interaction can be obtained between hyperthermia, cisplatin and radiation and that this interaction is more likely to occur in cell lines which are more sensitive to cisplatin.

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The effect of trimodality treatment consisting of hyperthermia, cisplatin and radiation was investigated in two cell lines with different sensitivities to cisplatin. Hyperthermia treatment was performed for 1 h at 41˚C and 43˚C in order to compare the effects of the two temperatures. Clonogenic assays were performed with cisplatin-sensitive SiHa human cervical carcinoma and cisplatin-resistant SW-1573 human lung carcinoma cell lines. Cells were treated with various combinations of hyperthermia, cisplatin and radiation. Radiation was performed after 1 h of simultaneous hyperthermia and cisplatin treatment. Cisplatin exposure was for 1 h or continuous without refreshment of the cisplatin-containing medium. SiHa cells were more sensitive to cisplatin than SW-1573 cells. Hyperthermia at 41˚C decreased survival in SW-1573 cells but was not cytotoxic in SiHa cells. Hyperthermia at 43˚C decreased survival dramatically in both cell lines with SiHa being the most sensitive. The addition of hyperthermia at 41˚C and 43˚C to cisplatin treatment led to enhanced cell kill in both cell lines compared with cisplatin alone. Radiosensitization was observed after continuous but not after 1 h of cisplatin treatment. Hyperthermia at 43˚C increased radiosensitivity whereas hyperthermia at 41˚C did not. A combination of 41˚C hyperthermia with continuous cisplatin treatment had an additive effect on SW-1573 cells but enhanced cisplatin radiosensitivity of SiHa cells. In SW-1573 cells trimodality treatment using 43˚C hyperthermia enhances cisplatin radiosensitivity. We conclude that hyperthermia at 43˚C enhances cisplatin-induced radiosensitization in both cisplatin-sensitive and-resistant cell lines. Hyper-thermia at 41˚C was also able to increase cisplatin-induced radiosensitivity but only in the cisplatin-sensitive SiHa cell line.