Objective. Boron neutron capture therapy is a cancer radiotherapy that uses the selective uptake of boron compounds by tumor cells, followed by neutron irradiation. Conventional dosimetry generally assumes a homogeneous boron distribution within tissues, yet evidence indicates intracellular heterogeneity. This work aims to improve the photon isoeffective dose model (PID) for glioblastoma multiforme (GBM) by incorporating subcellular-scale effects: (i) a correction factor for the stochastic nature of energy deposition due to intracellular boron localization, and (ii) the treatment of the nucleus-to-cytoplasm boron concentration ratio as a stochastic variable.Approach. The boron-10 microdistribution in U-87 glioblastoma cells was quantified for the first time through neutron autoradiography, revealing preferential accumulation in the nucleus. Following these experimental data, the nucleus-to-cytoplasm boron concentration ratio was described by a lognormal random variable, consistent with biological uptake processes. The correction factor was applied to the dosimetry of U-87 radiobiological data. Then, updated radiobiological parameters and subcellular-scale effects were integrated into the PID formalism and applied to a clinical case of GBM.Main results. The outcome was aMicrodosimetric PID, which extends conventional PID by explicitly including intracellular boron heterogeneity. Applied to U-87 data, proposed corrections revealed a 47% reduction in the compound biological effectiveness factor compared to conventional calculations, showing that neglecting subcellular distribution substantially overestimates the boron dose. For the clinical case, the total dose and 1 year progression-free survival (PFS) differed only by 4% and 3%, respectively, compared to conventional dosimetry. However, perturbation analyses indicated that under higher intracellular heterogeneity, plausiblein vivo, the deviations could become substantial (up to 22% in dose and 68% in PFS).Significance. These findings highlight the relevance of subcellular-scale modeling. The proposed microdosimetric model, grounded on experimentally derived microdosimetric corrections, provides a robust framework to improve both the accuracy and the personalization of BNCT treatment planning.

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Introduction:The ISOLPHARM project has the aim of developing novel radiopharmaceuticals using the wide choice of radionuclides produced by Isotope Separation OnLine (ISOL) at LNL-INFN in the SPES facility, which is currently nearing completion. One of the most promising candidates for Targeted Radionuclide Therapy (TRT) is the beta-emitting radiometal silver-111, obtainable carrier-free irradiating a uranium carbide target with a proton beam and applying the ISOL technique. Until SPES will become fully operational, small quantities of silver-111 are produced by the TRIGA Mark II nuclear reactor hosted by the LENA facility of the University of Pavia to begin the preclinical research. The present work concerns the first radiobiological experiment involving silver-111.Methods:Different activity concentrations of the aforementioned radiometal are administered to the UMR-106 rat osteosarcoma and LNCaP human prostate cancer cell lines through the culture medium. The survival curves after four and six days of exposure, as well as the recurrence of foci of DNA repair proteins and micronuclei, are evaluated as a function of the absorbed dose and compared to the control cultures. According to the MIRD formalism, a dosimetric analysis is performed taking advantage of cellular S-values simulated with the Monte Carlo code Geant4 in a generalized cell geometry. This makes it possible to relate the experimental outcome, namely the surviving cells after the exposure cycles, to the absorbed dose in the cell nucleus or in the whole cell environment.Results and discussionThe results show a difference in the response of the two cell lines, probably due to the thresholds of their DNA repair pathways, and highlight a possible weakness of the linear-quadratic model when applied to this kind of radiobiological studies.

Despite more than half of all oncological patients undergo X-ray radiotherapy (XRT), significant efforts are required to improve its efficacy against hypoxic tumor regions and, at the same time, to expand the therapeutic window to spare normal tissues. The use of radiosensitizers, the personalization of radiation dose planning aided by imaging with magnetic resonance imaging (MRI) and X-ray computed tomography (CT), and the implementation of boron neutron capture therapy (BNCT) are three strategies to encompass the limits of XRT. Here, these three strategies are leveraged by designing and achieving a theranostic platform based on trimetallic Au-Fe-B nanoparticles (NPs). According to density functional theory calculations, chemodegradable Au-Fe-B nanostructures are not achievable under thermodynamic equilibrium conditions. Hence, Au-Fe-B NPs were synthesized by laser ablation in liquid, because it is a nonequilibrium process, followed by a tailored cleaning protocol. The Au-Fe-B NPs were coated with biocompatible polymers and showed several useful properties for nanomedicine application, such as chemical degradation in a physiological environment, contrast ability for MRI and CT, in vitro radiosensitization efficacy for XRT and BNCT, and consistent intracellular uptake. These functionalities can enable advanced studies on tumor treatment with complementary therapeutic strategies guided by anatomic imaging, leading to more effective oncological protocols.

Boron Neutron Capture Therapy (BNCT) is a binary radiotherapy based on the intravenous administration of a borated drug to the patient and the subsequent irradiation with a low-energy neutron beam. The borated formulation accumulates in the tumor cells, and when neutrons interact with boron, a nuclear capture reaction occurs, releasing high-linear energy transfer, short-range particles that cause lethal damage to the cancer cells. Due to its selectivity, BNCT has the potential to treat aggressive brain tumors such as glioblastoma multiforme (GBM), minimizing the side effects. GBM is a brain neoplasia that poses significant treatment challenges due to its invasiveness and resistance to conventional treatments. This work aims to find a suitable model for calculating the photon isoeffective dose for GBM, producing ad hoc radiobiological data to feed the model. The dose-survival curves have been obtained with two different photon radiation sources as the reference with a thermal neutron beam and neutrons in the presence of boron. The fitted parameters have been obtained as the input for the photon isoeffective dose and the traditional RBE model. For the first time, the radiobiological parameters of a photon isoeffective dose model were produced for BNCT of GBM. Photon isoeffective dose value can differ up to 32% using gamma photons and low-energy x-rays. Photon isoeffective dose values are lower (17%) than the RBE model currently employed in clinical trials. The results highlight the impact of the reference radiation chosen for the isoeffective dose calculation and the importance of feeding the model with the appropriate radiobiological parameters.The dosimetry obtained with the new radiobiological data is consistent with the dose delivered in modern stereotactic radiotherapy, enabling tumor control predictions.

Boron neutron capture therapy (BNCT) is an oncological treatment based on the neutron capture reaction on 10B. The only two compounds approved for phase I/II clinical trials are sodium borocaptate (BSH) and 4-boronophenylalanine (BPA). While BPA has been widely exploited in clinical trials, the use of BSH is limited due to its insufficient uptake by tumor cells. Herein, we report a novel formulation based on silk fibroin nanoparticles (SFNs), capable of loading a large amount of borocaptate ions. The nanoparticles have been characterized and tested on U87 glioma cells, and boron uptake was measured using neutron autoradiography, which involved irradiating the samples in a thermal neutron field. Measurements demonstrated the crucial role of the nanocarrier in enhancing boron internalization. Notably, SFNs-BSH achieved 29.5 ppm total boron uptake in U87 cells - comparable to clinical BPA - at 4x lower dose.

BackgroundBoron Neutron Capture Therapy (BNCT) is a binary radiotherapy based on the intravenous administration of a borated drug to the patient and the subsequent irradiation with a low-energy neutron beam. The borated formulation accumulates in the tumor cells, and when neutrons interact with boron, a nuclear capture reaction occurs, releasing high-linear energy transfer, short-range particles that cause lethal damage to the cancer cells. Due to its selectivity, BNCT has the potential to treat aggressive brain tumors such as glioblastoma multiforme (GBM), minimizing the side effects. GBM is a brain neoplasia that poses significant treatment challenges due to its invasiveness and resistance to conventional treatments.PurposeThis work aims to find a suitable model for calculating the photon isoeffective dose for GBM, producing ad hoc radiobiological data to feed the model.1.2.3.4.5.MethodsTo describe adequately the dose-effect relation of BNCT for GBM, the following strategy has been applied We studied the impact of choosing two different photon radiation types (x- or gamma- rays) We assumed that the correct description of the photon-equivalent dose is obtained with the photon isoeffective dose model. This model calculates the photon dose that equals the cell survival obtained with BNCT, taking into account synergism and sub-lethal damage (SLD). Survival curves as a function of the dose for the human GBM U87 cell line were constructed using the clonogenic assays for irradiation with photons (reference), neutron beam, and BNCT. Survival curves were fitted with the modified linear quadratic model, using SLD repair times derived for U87. The radiobiological parameters were determined for the photon isoeffective dose model. The model was applied to a clinical case that received BNCT in Taiwan. Treatment planning has been simulated using an accelerator-based designed neutron beam following the real treatment process and parameters. The results were discussed and compared to the current method, which employs relative biological effectiveness (RBE) factors to obtain BNCT dosimetry in photon-equivalent units.ResultsThe dose-survival curves have been obtained with two different photon radiation sources as the reference with a thermal neutron beam and neutrons in the presence of boron. The fitted parameters have been obtained as the input for the photon isoeffective dose and the traditional RBE model. For the first time, the radiobiological parameters of a photon isoeffective dose model were produced for BNCT of GBM. Photon isoeffective dose value can differ up to 32% using gamma photons and low-energy x-rays. Photon isoeffective dose values are lower (17%) than the RBE model currently employed in clinical trials.ConclusionThe results highlight the impact of the reference radiation chosen for the isoeffective dose calculation and the importance of feeding the model with the appropriate radiobiological parameters.The dosimetry obtained with the new radiobiological data is consistent with the dose delivered in modern stereotactic radiotherapy, enabling tumor control predictions.

Background: Boron Neutron Capture Therapy (BNCT) is a promising cancer treatment that combines tumor-selective boron delivery agents with thermal neutrons to kill cancer cells while sparing normal tissue. BNCT requires boron-containing compounds that exhibit high tumor selectivity and achieve therapeutic boron concentrations within tumor cells. This work focuses on the early development of a novel boron cluster carbohydrate derivative based on the glucosamine structure. Our results indicate that this derivative may have advantages over the typical boron delivery agent used in clinical applications and may significantly improve boron delivery capacity at the cellular level. Methods: The performance of the compound in terms of boron uptake was tested in the U87-MG glioblastoma cell line employing neutron autoradiography imaging and quantification. Results: The compound was non-toxic for cells, and it showed a remarkable capacity to enrich cells with boron. The ratio between boron concentration provided in the culture medium and boron concentration achieved in cells was compared to that obtained with boronophenylalanine (BPA), the gold standard in BNCT. The result demonstrated a significantly better performance compared with BPA, showing that the novel agent can concentrate boron in cells more than in culture medium. Conclusions: The encouraging preliminary results provide a starting point for its potential application in in vivo tests.

Boron-Neutron Capture Therapy (BNCT) is a tumor-selective radiotherapy, based on the nuclear capture reaction 10B(n,α)7Li producing short range α-particles and recoiling 7Li nuclei exclusively confined to boron-enriched cancer cells. These particles possess high Linear Energy Transfer (LET) and mainly generate clustered DNA strand breaks, which are less faithfully restored by intracellular repair. Mis-rejoined breaks yield chromosome aberrations (CAs), which, for high-LET radiation, are more complex in nature than after sparsely ionizing photons/electrons used in conventional radiotherapy, which leads to increased cell-killing ability. However, such a radiobiological tenet of BNCT has been scantily studied at the DNA level. Therefore, the aim of this work was to evaluate CAs induced by BNCT in comparison to X-rays in genomically stable normal human epithelial mammary MCF10A cells. Two Fluorescence In Situ Hybridization (FISH)-based techniques were applied to calyculin A-induced prematurely condensed chromosomes: Whole Chromosome Painting and multicolor(m)-FISH. Not only did BNCT induce a greater CA frequency than X-ray irradiation, but m-FISH karyotype-wide analysis confirmed that CAs following BNCT exhibited a much higher degree of complexity compared to X-rays. To our knowledge, this is the first time that such evidence supporting the radiobiological superiority of BNCT has been shown.

Osteosarcoma is the most frequently primary malignant bone tumor characterized by infiltrative growth responsible for relapses and metastases. Treatment options are limited, and a new therapeutic option is required. Boron neutron capture therapy (BNCT) is an experimental alternative radiotherapy able to kill infiltrative tumor cells spearing surrounding healthy tissues. BNCT studies are performed on 2D in vitro models that are not able to reproduce pathological tumor tissue organization or on in vivo animal models that are expensive, time-consuming and must follow the 3R's principles. A 3D in vitro model is a solution to better recapitulate the complexity of solid tumors meanwhile limiting the animal's use. Objective of this study is to optimize the technical assessment for developing a 3D in vitro osteosarcoma model as a platform for BNCT studies: printing protocol, biomaterial selection, cell density, and crosslinking process. The best parameters that allow a fully colonized 3D bioprinted construct by rat osteosarcoma cell line UMR-106 are 6 × 106 cells/ml of hydrogel and 1% CaCl2 as a crosslinking agent. The proposed model could be an alternative or a parallel approach to 2D in vitro culture and in vivo animal models for BNCT experimental study.

Three-dimensional (3D) bioprinting allows the production of artificial 3D cellular microenvironments thanks to the controlled spatial deposition of bioinks. Proper bioink characterization is required to achieve the essential characteristics of printability and biocompatibility for 3D bioprinting. In this work, a protocol to standardize the experimental characterization of a new bioink is proposed. A functionalized hydrogel based on gelatin and chitosan was used. The protocol was divided into three steps: pre-printing, 3D bioprinting, and post-printing. For the pre-printing step, the hydrogel formulation and its repeatability were evaluated. For the 3D-bioprinting step, the hydrogel-printability performance was assessed through qualitative and quantitative tests. Finally, for the post-printing step, the hydrogel biocompatibility was investigated using UMR-106 cells. The hydrogel was suitable for printing grids with good resolution from 4 h after the cross-linker addition. To guarantee a constant printing pressure, it was necessary to set the extruder to 37 °C. Furthermore, the hydrogel was shown to be a valid biomaterial for the UMR-106 cells' growth. However, fragmentation of the constructs appeared after 14 days, probably due to the negative osteosarcoma-cell interference. The protocol that we describe here denotes a strong approach to bioink characterization to improve standardization for future biomaterial screening and development.

Neutron capture therapy (NCT) is a form of radiotherapy that exploits the potential of some specific isotopes to capture thermal neutrons and subsequently yield high linear energy transfer (LET) particles, suitable for cancer treatment. Recently, relevant technological improvements have been made in terms of accelerators as suitable neutron sources for NCT at hospitals. However, low selective delivery of current drugs to cancer cells remains as the main challenge for successful clinical application of NCT. This work presents an innovative and previously unexplored approach for the design of nanotherapeutic NCT agents. Herein, a new concept based on carbon nanomaterials that seal 6Li active NCT nuclides is investigated. The 6Li active species are located in the inner cavity of the nanocarrier (carbon nanohorns or carbon nanotubes) and therefore, completely protected from the biological environment, avoiding toxicity and degradation. After encapsulation of the active cargo, the external surface of the nanocarrier is modified for improved biocompatibility. The developed 6Li-filled carbon nanohorns offered the possibility to explore 6Li compounds as active NCT agents by delivering therapeutic doses to cancer cells. We envisage that nanoencapsulation of 6Li can trigger the successful development and implementation of Lithium Neutron Cancer Therapy (LiNCT).

New multifunctional theranostic vectors allow the expansion of cancer therapeutic approaches toward scarcely investigated fields. One example is the combination of boron neutron capture therapy (BNCT) and X-ray radiotherapy (XRT) for treating normal and XRT-resistant hypoxic tumor regions and reduce recurrence. Of great relevance for BNCT is also the support of viable, rapid, safe, and reliable techniques for the localization and quantification of the radiosensitizers in the tissues. To address these challenges, polymer-coated Au-B nanoparticles (NPs) are obtained starting from a laser ablation in liquid process. Despite thermodynamic constraints, the two elements coexist by short-range boron segregation in the gold lattice, as demonstrated experimentally and explained with the support of density functional theory calculations. Thus, the Au-B NPs maintain a marked gold character such as biocompatibility, stability, and straightforward surface chemistry with thiolated compounds, desirable for the integration with agents capable of cell targeting and internalization. Overall, the Au-B NPs exhibit the appropriate features for the investigation of combined BNCT and XRT, supported by the localization and quantification with X-ray computed tomography imaging. Besides, the Au-B nanotechnology tool is achievable without renouncing to reproducibility, environmental sustainability, and cost affordability thanks to the laser-assisted synthetic pathway.

Boron neutron capture therapy (BNCT) is a treatment modality for cancer that involves radiations of different qualities. A formalism that proved suitable to compute doses in photon-equivalent units is the photon isoeffective dose model. This study addresses the question whether considering in vitro or in vivo radiobiological studies to determine the parameters involved in photon isoeffective dose calculations affects the consistency of the model predictions. The analysis is focused on head and neck squamous cell carcinomas (HNSCC), a main target that proved to respond to BNCT. The photon isoeffective dose model for HNSCC with parameters from in vitro studies using the primary human cell line UT-SCC-16A was introduced and compared to the one previously reported with parameters from an in vivo oral cancer model in rodents. Both models were first compared in a simple scenario by means of tumor dose and control probability calculations. Then, the clinical impact of the different dose models was assessed from the analysis of a group of squamous cell carcinomas (SCC) patients treated with BNCT. Traditional dose calculations using the relative biological effectiveness factors derived from the SCC cell line were also analyzed. Predictions of tumor control from the evaluated models were compared to the patients' outcome. The quantification of the biological effectiveness of the different radiations revealed that relative biological effectiveness/compound biological effectiveness (RBE/CBE) factors for the SCC cell line are up to 20% higher than those assumed in clinical BNCT, highlighting the importance of using experimental data intimately linked to the tumor type to derive the model's parameters. The comparison of the different models showed that photon isoeffective doses based on in vitro data are generally greater than those from in vivo data (∼8-16% for total tumor absorbed doses of 10-15 Gy). However, the predictive power of the two models was not affected by these differences: both models fulfilled conditions to guarantee a good predictive performance and gave predictions statistically compatible with the clinical outcome. On the other hand, doses computed with the traditional model were substantially larger than those obtained with both photon isoeffective models. Moreover, the traditional model is statistically rejected, which reinforces the assertion that its inconsistencies are intrinsic and not due to the use of RBE/CBE factors obtained for a tumor type different from HN cancer. The results suggest that the nature of the radiobiological data would not affect the consistency of the photon isoeffective dose model in the studied cases of SCC head and neck cancer treated with BPA-based BNCT.

Purpose: One of the obstacles to the application of Boron Neutron Capture Therapy (BNCT) and Proton Boron Fusion Therapy (PBFT) concerns the measurement of borated carriers' biodistribution. The objective of the present study was to evaluate the in vitro internalization of the F-19-labelled p-boronophenylalanine (F-19-BPA) in the human cancer pancreatic cell line (PANC-1) for the potential application of BNCT and PBFT in pancreatic cancer. The F-19-BPA carrier has the advantage that its bio-distribution may be monitored in vivo using F-19-Nuclear Magnetic Resonance (F-19 NMR). Materials and methods: The F-19-BPA internalization in PANC-1 cells was evaluated using three independent techniques on cellular samples left in contact with growing medium enriched with 13.6 mM F-19-BPA corresponding to a B-11 concentration of 120 ppm: neutron autoradiography, which quantifies boron; liquid chromatography hyphenated to tandem mass spectrometry and UV-Diode Array Detection (UV-DAD), which quantifies F-19-BPA molecule; and F-19 NMR spectroscopy, which detects fluorine nuclei. Results: Our studies suggested that F-19-BPA is internalized by PANC-1 cells. The three methods provided consistent results of about 50% internalization fraction at 120 ppm of B-11. Small variations (less than 15%) in internalization fraction are mainly dependent on the proliferation state of the cells. Conclusions: The ability of F-19 NMR spectroscopy to study F-19-BPA internalization was validated by well-established independent techniques. The multimodal approach we used suggests F-19-BPA as a promising BNCT/PBFT carrier for the treatment of pancreatic cancer. Since the quantification is performed at doses useful for BNCT/PBFT, F-19 NMR can be envisaged to monitor F-19-BPA bio-distribution during the therapy.

Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. Methods: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. Results: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse model; in the zebrafish model, we also demonstrated the downregulation of tumor neo-angiogenesis, and in the orthotopic rat model, we observed impaired N1-S1 grafting in a healthy liver. Conclusion: We demonstrate for the first time that 5-Aza can impair HCC development via upregulation of miR-139-5p, which in turn impairs the ROCK2/cyclin D1/E2F1/cyclin B1 pro-proliferative pathway and the ROCK2/MMP-2 pro-migratory pathway. Thus, we provide novel information about 5-Aza mechanisms of action and deepen the knowledge about the crosstalk among ROCK2/cyclin D1/E2F1/cyclin B1/p27kip1/MMP-2 in HCC.

The study aims at using the bioprinting technique to create an in vitro 3D construct of osteosarcoma, as an alternative model for studies related to Boron Neutron Capture Therapy (BNCT).