The common experimental use of B16-F10 melanoma cells focuses on exploring their metastatic potential following intravenous injection into mice. In this study, B16-F10 cells are used to develop a primary tumor model by implanting them directly into the ears of C57BL/6J mice. The model represents a reproducible and easily traceable tool for local tumor growth and for making additional in vivo observations, due to the localization of the tumors. This model is relatively simple and involves (i) surgical opening of the ear skin, (ii) removal of a square-piece of cartilage followed by (iii) the implantation of tumor cells with fibrin gel. The remodeling of the fibrin gel within the cartilage chamber, accompanying tumor proliferation, results in the formation of blood vessels, lymphatics and tissue matrix that can be readily distinguished from the pre-existing skin structures. Moreover, this method avoids the injection-enforced artificial spread of cells into the pre-existing lymphatic vessels. The tumors have a highly reproducible exponential growth pattern with a tumor doubling time of around 1.8 days, reaching an average volume of 85mm³ 16 days after implantation. The melanomas are densely cellular with proliferative indices of between 60 and 80%. The induced angiogenesis and lymphangiogenesis resulted in the development of well-vascularized tumors. Different populations of immunologically active cells were also present in the tumor; the population of macrophages decreases with time while the population of T cells remained quasi constant. The B16-F10 tumors in the ear frequently metastasized to the cervical lymph nodes, reaching an incidence of 75% by day 16. This newly introduced B16-F10 melanoma model in the ear is a powerful tool that provides a new opportunity to study the local tumor growth and metastasis, the associated angiogenesis, lymphangiogenesis and tumor immune responses. It could potentially be used to test different treatment strategies.

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.

Background: Microbeam Radiation Therapy (MRT) is an innovative approach in radiation oncology where a collimator subdivides the homogeneous radiation field into an array of co-planar, high-dose beams which are tens of micrometres wide and separated by a few hundred micrometres. Objective: This scoping review was conducted to map the available evidence and provide a comprehensive overview of the similarities, differences, and outcomes of all experiments that have employed animal models in MRT. Methods: We considered articles that employed animal models for the purpose of studying the effects of MRT. We searched in seven databases for published and unpublished literature. Two independent reviewers screened citations for inclusion. Data extraction was done by three reviewers. Results: After screening 5688 citations and 159 full-text papers, 95 articles were included, of which 72 were experimental articles. Here we present the animal models and pre-clinical radiation parameters employed in the existing MRT literature according to their use in cancer treatment, non-neoplastic diseases, or normal tissue studies. Conclusions: The study of MRT is concentrated in brain-related diseases performed mostly in rat models. An appropriate comparison between MRT and conventional radiotherapy (instead of synchrotron broad beam) is needed. Recommendations are provided for future studies involving MRT.

Background: Synchrotron Microbeam Radiotherapy (MRT) significantly improves local tumour control with minimal normal tissue toxicity. MRT delivers orthovoltage X-rays at an ultra-high “FLASH” dose rate in spatially fractionated beams, typically only few tens of micrometres wide. One of the biggest challenges in translating MRT to the clinic is its use of high peak doses, of around 300–600 Gy, which can currently only be delivered by synchrotron facilities. Therefore, in an effort to improve the translation of MRT to the clinic, this work studied whether the temporal fractionation of traditional MRT into several sessions with lower, more clinically feasible, peak doses could still maintain local tumour control. Methods: Two groups of twelve C57Bl/6J female mice harbouring B16-F10 melanomas in their ears were treated with microbeams of 50 µm in width spaced by 200 µm from their centres. The treatment modality was either (i) a single MRT session of 401.23 Gy peak dose (7.40 Gy valley dose, i.e., dose between beams), or (ii) three MRT sessions of 133.41 Gy peak dose (2.46 Gy valley dose) delivered over 3 days in different anatomical planes, which intersected at 45 degrees. The mean dose rate was 12,750 Gy/s, with exposure times between 34.2 and 11.4 ms, respectively. Results: Temporally fractionated MRT ablated 50% of B16-F10 mouse melanomas, preventing organ metastases and local tumour recurrence for 18 months. In the rest of the animals, the median survival increased by 2.5-fold in comparison to the single MRT session and by 4.1-fold with respect to untreated mice. Conclusions: Temporally fractionating MRT with lower peak doses not only maintained tumour control, but also increased the efficacy of this technique. These results demonstrate that the solution to making MRT more clinically feasible is to irradiate with several fractions of intersecting arrays with lower peak doses. This provides alternatives to synchrotron sources where future microbeam radiotherapy could be delivered with less intense radiation sources.

Radiation therapy (RT) is a vital component of multimodal cancer treatment, and its immunomodulatory effects are a major focus of current therapeutic strategies. Macrophages are some of the first cells recruited to sites of radiation-induced injury where they can aid in tissue repair, propagate radiation-induced fibrogenesis and influence tumour dynamics. Microbeam radiation therapy (MRT) is a unique, spatially fractionated radiation modality that has demonstrated exceptional tumour control and reduction in normal tissue toxicity, including fibrosis. We conducted a morphological analysis of MRT-irradiated normal liver, lung and skin tissues as well as lung and melanoma tumours. MRT induced distinct patterns of DNA damage, reflecting the geometry of the microbeam array. Macrophages infiltrated these regions of peak dose deposition at variable timepoints post-irradiation depending on the tissue type. In normal liver and lung tissue, macrophages clearly demarcated the beam path by 48 h and 7 days post-irradiation, respectively. This was not reflected, however, in normal skin tissue, despite clear DNA damage marking the beam path. Persistent DNA damage was observed in MRT-irradiated lung carcinoma, with an accompanying geometry-specific influx of mixed M1/M2-like macrophage populations. These data indicate the unique potential of MRT as a tool to induce a remarkable accumulation of macrophages in an organ/tissue-specific manner. Further characterization of these macrophage populations is warranted to identify their organ-specific roles in normal tissue sparing and anti-tumour responses.

Despite the recent progress, current treatment modalities are not able to eradicate cancer. We show that Microbeam Radiotherapy (MRT), an innovative type of Spatially Fractionated Radiotherapy, can control murine melanoma by activating the host's own immune system. The beneficial effects are very pronounced in comparison to uniform radiotherapy traditionally employed in the clinic. Our results show that MRT increased antigen presentation, activating Cytotoxic T Lymphocytes (CTLs) which are essential to MRT's treatment efficacy in melanoma. Depletion of CTLs abrogated treatment response. Multiplex nucleic acid hybridization technology revealed key features of lymphocyte populations such as proliferation, differentiation, and ligand-receptor interactions. In addition, CTLs were shown to be essential for locoregional metastatic control and systemic abscopal effects confirmed by activation of antigen presenting cells and CTL trafficking in the tumour-draining lymph nodes. MRT also showed a synergistic effect with immunotherapy. Overall, MRT induces a robust antitumour immune response, acting like an in situ vaccination, which could be exploited to treat a variety of treatment-resistant malignancies.