Yolanda Prezado

Particle minibeam therapy (PMBT) has emerged as an advanced modality within spatially fractionated radiation therapy, using the physical properties of protons, ions, and electrons to achieve superior normal tissue sparing whilst preserving spatial dose distribution at depths that are unachievable in conventional kV or MV X‐rays. As with any novel radiotherapy technique, successful translation into routine clinical practice requires robust evidence of efficacy, safety, and technical feasibility. This review presents a coordinated roadmap for the development and future implementation of PMBT, identifying the key challenges that must be addressed to enable safe and effective clinical implementation. This review highlights the interconnected nature of PMBT research and proposes the priority areas for future investigation, and the steps necessary to guide PMBT from experimental concept, towards clinical implementation. As most of the steps are relevant to all particle types, PMBT will be referred to in general, where specific particle types will be discussed where appropriate.

Minibeam radiotherapy (MBRT) is a form of spatially fractionated radiation therapy that uses arrays of submillimetric beamlets to generate highly heterogeneous dose distributions composed of alternating high-dose peaks and low-dose valleys. A growing body of preclinical evidence demonstrates that MBRT can maintain, and in some cases improve, tumor control while markedly reducing normal tissue toxicity. Beyond its physical characteristics, MBRT induces distinct biological responses, including vascular modulation, immune activation, redox signaling, and intercellular communication, supporting its characterization as a biologically distinct irradiation paradigm rather than a simple dosimetric optimization strategy. Despite these promising findings, important challenges remain for mainstream clinical implementation. Conventional dosimetry protocols and treatment planning systems are not designed to resolve submillimetric dose patterns, requiring high-resolution detectors, biologically meaningful dose metrics, and Monte-Carlo-based planning approaches. In addition, inconsistent definitions of key parameters such as peak dose, valley dose, and peak-to-valley dose ratio (PVDR) limit reproducibility and comparison across studies. Considerations related to motion and delivery accuracy may also become relevant in certain applications, particularly for extracranial treatments, where preservation of the intended spatial dose modulation may influence therapeutic outcomes. Recent veterinary studies, early human applications, and expanding preclinical evidence continue to support MBRT as a promising strategy for widening the therapeutic window of radiotherapy.

Purpose. Spatially fractionated radiation therapy (SFRT) enables the delivery of high peak doses while sparing normal tissue, yet the relative contributions of peak dose, valley dose and spatial dose distribution remain unclear. This study investigates whether spatial dose organization contributes to normal tissue response beyond conventional dosimetry descriptors. Methods. Acute and late skin responses were evaluated in a murine hind-limb model after single-fraction irradiation with clinically pertinent Mini-GRID and planar beam configurations that allow for peak and valley dose effects to be decoupled. Longitudinal clinical scoring, histopathological evaluation and quantitative proteomic analysis were performed up to 90 d following irradiation. Results. With the peak dose held constant, Mini-GRID irradiation resulted in reduced toxicity compared with planar irradiation delivering higher valley doses. In contrast, conditions with similar valley doses but substantially different peak doses showed comparable outcomes. These findings suggest that maximal dose alone does not act as a predictor of normal tissue response. Distinct molecular signatures associated with spatial dose heterogeneity were identified through a proteomic analysis corresponding to spatial dose heterogeneity, which was consistent with regulated tissue remodeling and resolution of inflammation. Conclusion. These findings suggest that spatial dose distribution contributes significantly to normal tissue response beyond peak and valley dose magnitude alone. While valley dose defines baseline tissue injury, spatial dose organization modulates repair capacity and long-term outcome. This work provides a radiobiological framework for SFRT optimization and supports the incorporation of spatial dose descriptors, beyond conventional metrics such as PVDR, into treatment design.

Partial tumor irradiation targeting hypoxic/hypovascularized/hypometabolic tumor segment (PATHY) while sparing the peritumoral immune microenvironment has emerged as an innovative radiotherapy strategy designed to exploit non-targeted and immune-mediated effects. Although promising clinical responses have been reported, the limited availability and technical complexity of preclinical PATHY implementations have constrained mechanistic investigation and rational optimization of treatment parameters. Here, we establish a PATHY-inspired partial tumor irradiation (PTI) workflow on a Small Animal Radiation Research Platform (SARRP) and evaluate its therapeutic efficacy in a rat glioma model. A protocol was developed integrating MRI-based target delineation, millimetric small-field dosimetry, and in vivo quality assurance to selectively irradiate the tumor core using orthovoltage photons. Tumor-bearing rats were assigned to one of three PTI regimens differing in temporal fractionation-conventional fractionation (15 Gy × 3, Δt = 24 h), accelerated fractionation (15 Gy × 3, Δt = 8 h), or single-fraction PTI (30 Gy × 1)-or to a non-irradiated control group. All PTI regimens significantly prolonged overall survival compared with controls, yielding median survival increases ranging from 32% to 62%. No statistically significant differences were observed among the irradiated groups, indicating that, under the investigated dose and fractionation conditions, interfraction timing did not significantly influence survival outcome in this highly radioresistant and immunosuppressive glioma model. This study establishes a robust and reproducible preclinical framework for PTI and provides the first in vivo evidence of its therapeutic potential in glioma. By enabling systematic investigation of partial tumor high-dose irradiation, this platform lays the groundwork for future studies incorporating hypoxia-guided targeting and immune mechanistic endpoints to refine and translate PATHY-based radiotherapy strategies.

Minibeam Radiation Therapy (MBRT) uses sub-millimetric beams to generate highly heterogeneous dose distributions and has shown promising results in preclinical glioblastoma by preserving normal tissue while eliciting immune responses. However, the initiating molecular mechanisms remain unclear. We hypothesised that immunogenic cell death or cGAS-STING pathway activation could be involved. We evaluated cell death, damage-associated molecular patterns (calreticulin exposure and HMGB1 release), RT1a exposure, and cGAS-STING activation 24 h after irradiation. MBRT was compared with conventional radiation therapy (CRT; X-rays or protons) in glioblastoma cell lines (F98, RG2) and normal astrocytes using flow cytometry and qPCR. CRT induced dose-dependent tumour cell death, whereas MBRT caused lower, non-dose-dependent effects. Astrocytes showed similar responses to both modalities. Immunogenic markers were limited: CRT increased calreticulin and RT1a exposure more than MBRT, while HMGB1 was undetectable. No transcriptional activation of the cGAS-STING pathway was detected, although interferon-stimulated genes were partially upregulated in F98 cells. RG2 cells showed no immune activation. CRT selectively induced Cxcl10 expression in astrocytes. MBRT and CRT induce distinct biological and immune-related responses in tumour cells and astrocytes in vitro, suggesting different tumour-intrinsic mechanisms; whether these translate to broader immunogenic effects in vivo remains to be determined.

Spatially fractionated radiation therapy, particularly minibeam radiation therapy (MBRT), improves normal tissue sparing while maintaining tumor control compared with conventional radiotherapy (CRT). However, the mechanisms underlying these advantages remain unclear. EVs carrying miRNAs are increasingly recognized as mediators of radiation-induced intercellular communication and may contribute to non-targeted and cohort effects. We investigated the impact of MBRT versus CRT on EV-miRNA profiles in glioma and normal astrocyte cell lines. Rat glioma (RG2, F98) and astrocyte (CTX-TNA2) cell lines were irradiated with X-ray CRT or MBRT. Small EVs were isolated 24 h post-irradiation, characterized by transmission electron microscopy and nanoflow cytometry, and analyzed by small RNA sequencing. EV morphology, size, and expression of canonical markers (CD9, CD63, CD81) were preserved across conditions, indicating that irradiation primarily altered EV cargo. Glioma cells showed limited and cell-dependent changes. In RG2 cells, CRT increased miR-6319 and miR-374-5p, whereas MBRT modulated miR-151-5p in a dose-dependent manner and reduced miR-466c-5p at 5 Gy. No significant changes were observed in F98 cells. In contrast, astrocytes exhibited marked remodeling of EV-miRNA cargo after irradiation, including enrichment of miRNAs associated with stress adaptation, hypoxia-related signalling, and immune regulation, together with consistent depletion of miR-122b and miR-486 across irradiation conditions. MBRT and CRT were associated with differential remodeling of EV miRNA cargo, with a markedly stronger response in astrocytes than in glioma cells. These findings suggest altered EV signaling potential following irradiation and provide an initial molecular framework for investigating how spatial dose modulation may influence extracellular communication. Further functional validation in human-relevant systems is required to determine mechanistic and translational significance.