HollandPTC is an independent outpatient center for proton therapy, scientific research, and education. Patients with different types of cancer are treated with Intensity Modulated Proton Therapy (IMPT). Additionally, the HollandPTC R&D consortium conducts scientific research into the added value and improvements of proton therapy. To this end, HollandPTC created clinical and pre-clinical research facilities including a versatile R&D proton beamline for various types of biologically and technologically oriented research. In this work, we present the characterization of the R&D proton beamline of HollandPTC. Its pencil beam mimics the one used for clinical IMPT, with energy ranging from 70 up to 240 MeV, which has been characterized in terms of shape, size, and intensity. For experiments that need a uniform field in depth and lateral directions, a dual ring passive scattering system has been designed, built, and characterized. With this system, field sizes between 2 × 2 cm² and 20 × 20 cm² with 98 % uniformity are produced with dose rates ranging from 0.5 Gy/min up to 9 Gy/min. The unique and customized support of the dual ring system allows switching between a pencil beam and a large field in a very simple and fast way, making the HollandPTC R&D proton beam able to support a wide range of different applications.

A Geant4 based simulation platform of the Holland Proton Therapy Centre (HollandPTC, Netherlands) R&D beamline (G4HPTC-R&D) was developed to enable the planning, optimisation and advanced dosimetry for radiobiological studies. It implemented a six parameter non-symmetrical Gaussian pencil beam surrogate model to simulate the R&D beamline in both a pencil beam and passively scattered field configuration. Three different experimental proton datasets (70 MeV, 150 MeV, and 240 MeV) of the pencil beam envelope evolution in free air and depth-dose profiles in water were used to develop a set of individual parameter surrogate functions to enable the modelling of the non-symmetrical Gaussian pencil beam properties with only the ProBeam isochronous cyclotron mean extraction proton energy as input. This refined beam model was then benchmarked with respect to three independent experimental datasets of the R&D beamline operating in both a pencil beam configuration at 120 and 200 MeV, and passively scattered field configuration at 150 MeV. It was shown that the G4HPTC-R&D simulation platform can reproduce the pencil beam envelope evolution in free air and depth-dose profiles to within an accuracy on the order of ±5% for all tested energies, and that it was able to reproduce the 150 MeV passively scattered field to the specifications need for clinical and radiobiological applications.

Particle therapy (PT) used for cancer treatment can spare healthy tissue and reduce treatment toxicity. However, full exploitation of the dosimetric advantages of PT is not yet possible due to range uncertainties, warranting development of range-monitoring techniques. This study proposes a novel range-monitoring technique introducing the yet unexplored concept of simultaneous detection and imaging of fast neutrons and prompt-gamma rays produced in beam-tissue interactions. A quasi-monolithic organic detector array is proposed, and its feasibility for detecting range shifts in the context of proton therapy is explored through Monte Carlo simulations of realistic patient models and detector resolution effects. The results indicate that range shifts of 1mm can be detected at relatively low proton intensities (22.30 (13) × 10 ⁷ protons/spot) when spatial information obtained through imaging of both particle species are used simultaneously. This study lays the foundation for multi-particle detection and imaging systems in the context of range verification in PT.

Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. While X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can damage cancer cells while sparing the surrounding healthy tissue. However, the effects of protons on in vitro GBM models at the cellular level, especially when co-cultured with endothelial cells, the building blocks of brain micro-vessels, are still unexplored. In this work, novel 3D-engineered scaffolds inspired by the geometry of brain microvasculature are designed, where GBM cells cluster and proliferate. The architectures are fabricated by two-photon polymerization (2PP), pre-cultured with endothelial cells (HUVECs), and then cultured with a human GBM cell line (U251). The micro-vessel structures enable GBM in vivo-like morphologies, and the results show a higher DNA double-strand breakage in GBM monoculture samples when compared to the U251/HUVECs co-culture, with cells in 2D featuring a larger number of DNA damage foci when compared to cells in 3D. The discrepancy in terms of proton radiation response indicates a difference in the radioresistance of the GBM cells mediated by the presence of HUVECs and the possible induction of stemness features that contribute to radioresistance and improved DNA repair.

Glioblastoma (GBM) is a devastating cancer of the brain with an extremely poor prognosis. For this reason, besides clinical and preclinical studies, novel in vitro models for the assessment of cancer response to drugs and radiation are being developed. In such context, three-dimensional (3D)-engineered cellular microenvironments, compared to unrealistic two-dimensional (2D) monolayer cell culture, provide a model closer to the in vivo configuration. Concerning cancer treatment, while X-ray radiotherapy and chemotherapy remain the current standard, proton beam therapy is an appealing alternative as protons can be efficiently targeted to destroy cancer cells while sparing the surrounding healthy tissue. However, despite the treatment's compelling biological and medical rationale, little is known about the effects of protons on GBM at the cellular level. In this work, we designed novel 3D-engineered scaffolds inspired by the geometry of brain blood vessels, which cover a vital role in the colonization mechanisms of GBM cells. The architectures were fabricated by two-photon polymerization (2PP), cultured with U-251 GBM cells and integrated for the first time in the context of proton radiation experiments to assess their response to treatment. We employed Gamma H2A.X as a fluorescent biomarker to identify the DNA damage induced in the cells by proton beams. The results show a higher DNA double-strand breakage in 2D cell monolayers as compared to cells cultured in 3D. The discrepancy in terms of proton radiation response could indicate a difference in the radioresistance of the GBM cells or in the rate of repair kinetics between 2D cell monolayers and 3D cell networks. Thus, these biomimetic-engineered 3D scaffolds pave the way for the realization of a benchmark tool that can be used to routinely assess the effects of proton therapy on 3D GBM cell networks and other types of cancer cells.

Superheated nanodroplet (ND) vaporization by proton radiation was recently demonstrated, opening the door to ultrasound-based in vivo proton range verification. However, at body temperature and physiological pressures, perfluorobutane nanodroplets (PFB-NDs), which offer a good compromise between stability and radiation sensitivity, are not directly sensitive to primary protons. Instead, they are vaporized by infrequent secondary particles, which limits the precision for range verification. The radiation-induced vaporization threshold (i.e., sensitization threshold) can be reduced by lowering the pressure in the droplet such that ND vaporization by primary protons can occur. Here, we propose to use an acoustic field to modulate the pressure, intermittently lowering the proton sensitization threshold of PFB-NDs during the rarefactional phase of the ultrasound wave. Simultaneous proton irradiation and sonication with a 1.1 MHz focused transducer, using increasing peak negative pressures (PNPs), were applied on a dilution of PFB-NDs flowing in a tube, while vaporization was acoustically monitored with a linear array. Sensitization to primary protons was achieved at temperatures between 29 °C and 40 °C using acoustic PNPs of relatively low amplitude (from 800 to 200 kPa, respectively), while sonication alone did not lead to ND vaporization at those PNPs. Sensitization was also measured at the clinically relevant body temperature (i.e., 37 °C) using a PNP of 400 kPa. These findings confirm that acoustic modulation lowers the sensitization threshold of superheated NDs, enabling a direct proton response at body temperature.

The potential of proton therapy to improve the conformity of the delivered dose to the tumor volume is currently limited by range uncertainties. Injectable superheated nanodroplets have recently been proposed for ultrasound-based in vivo range verification, as these vaporize into echogenic microbubbles on proton irradiation. In previous studies, offline ultrasound images of phantoms with dispersed nanodroplets were acquired after irradiation, relating the induced vaporization profiles to the proton range. However, the aforementioned method did not enable the counting of individual vaporization events, and offline imaging cannot provide real-time feedback. In this study, we overcame these limitations using high-frame-rate ultrasound imaging with a linear array during proton irradiation of phantoms with dispersed perfluorobutane nanodroplets at 37°C and 50°C. Differential image analysis of subsequent frames allowed us to count individual vaporization events and to localize them with a resolution beyond the ultrasound diffraction limit, enabling spatial and temporal quantification of the interaction between ionizing radiation and nanodroplets. Vaporization maps were found to accurately correlate with the stopping distribution of protons (at 50°C) or secondary particles (at both temperatures). Furthermore, a linear relationship between the vaporization count and the number of incoming protons was observed. These results indicate the potential of real-time high-frame-rate contrast-enhanced ultrasound imaging for proton range verification and dosimetry.