Microbeam Radiotherapy-A Novel Therapeutic Approach to Overcome Radioresistance and Enhance Anti-Tumour Response in Melanoma.

Melanoma is the deadliest type of skin cancer, due to its invasiveness and limited treatment efficacy. The main therapy for primary melanoma and solitary organ metastases is wide excision. Adjuvant therapy, such as chemotherapy and targeted therapies are mainly used for disseminated disease. Radiotherapy (RT) is a powerful treatment option used in more than 50% of cancer patients, however, conventional RT alone is unable to eradicate melanoma. Its general radioresistance is attributed to overexpression of repair genes in combination with cascades of biochemical repair mechanisms. A novel sophisticated technique based on synchrotron-generated, spatially fractionated RT, called Microbeam Radiation Therapy (MRT), has been shown to overcome these treatment limitations by allowing increased dose delivery. With MRT, a collimator subdivides the homogeneous radiation field into an array of co-planar, high-dose microbeams that are tens of micrometres wide and spaced a few hundred micrometres apart. Different preclinical models demonstrated that MRT has the potential to completely ablate tumours, or significantly improve tumour control while dramatically reducing normal tissue toxicity. Here, we discuss the role of conventional RT-induced immunity and the potential for MRT to enhance local and systemic anti-tumour immune responses. Comparative gene expression analysis from preclinical tumour models indicated a specific gene signature for an 'MRT-induced immune effect'. This focused review highlights the potential of MRT to overcome the inherent radioresistance of melanoma which could be further enhanced for future clinical use with combined treatment strategies, in particular, immunotherapy.

Gradual in vitro Evolution of Cefepime Resistance in an ST131 Escherichia coli Strain Expressing a Plasmid-Encoded CMY-2 ß-Lactamase.

BACKGROUND: In a previous report, a clinical ST131 Escherichia coli isolate (Ec-1),producing a plasmid-encoded AmpC ß-lactamase CMY-2, evolved in vivo under cefepime (FEP) treatment to the FEP-resistant Ec-2 strain expressing an extended-spectrum ß-lactamase CMY-33. To compare factors responsible for in vitro and in vivo FEP resistance, we reproduced in vitro FEP resistance evolution in Ec-1. METHODS: FEP-resistant mutants were generated by subjecting Ec-1 (FEP MIC = 0.125 mg/L) to sub-inhibitory concentrations of FEP. MICs were obtained by broth microdilution or Etest. Strains were sequenced on an Illumina HiSeq platform. Transcriptional levels and plasmid copy numbers were determined by real-time PCR. Outer membrane proteins (OMPs) were extracted and separated by SDS-PAGE. Growth kinetics was evaluated by measuring OD450. RESULTS: The CMY-2 expressed by Ec-1 evolved to a CMY-69 (strain EC-4) by an Ala294Pro substitution after 24 passages. After 30 passages, the FEP MIC increased to 256 mg/L (strain EC-32). SDS PAGE did not reveal any lack of OMPs in the mutant strains. However, bla CMY transcription levels were up to 14-times higher than in Ec-1, which was partially explained by mutations in the upstream region of repA resulting in a higher copy number of the bla CMY-harboring IncI1 plasmid. All mutants showed a slight growth defect but no significant difference in relative growth rates compared to Ec-1. CONCLUSION: In vitro sub-inhibitory concentrations of FEP resulted in the selection of resistance mutations altering the H-10 helix of the CMY-2 and increasing the plasmid copy number. Appropriate dosing strategies may help preventing resistance evolution during treatments.