Increased cytotoxic effects via combined treatment of magnetic field and radiation therapy in patient-derived organoids of pancreatic ductal adenocarcinoma

Increased cytotoxic effects via combined treatment of magnetic field and radiation therapy in patient-derived organoids of pancreatic ductal adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Increased cytotoxic effects via combined treatment of magnetic field and radiation therapy in patient-derived organoids of pancreatic ductal adenocarcinoma Luca Boldrini, Carlo Gugliemo Cattaneo, Lorenzo Placidi, Alessandra Ciucci, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9009316/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose/Objective Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with limited treatment options. Magnetic Resonance-guided Radiotherapy (MRIgRT) combines real-time imaging and irradiation under a static magnetic field (SMF), potentially influencing biological responses beyond its technical advantages. This study aimed to investigate the combined effects of SMF and radiotherapy on patient-derived PDAC organoids (PDOs) to assess whether MRI guidance modulates radiation-induced cytotoxicity. Material/Methods Two PDO lines (PDAC-9 and PDAC-12) were established from treatment-naïve PDAC tissues and cultured in 3D BME matrices. Organoids were exposed to three experimental conditions: (i) untreated control (CTRL), (ii) 10 Gy irradiation using a standard linac (IR), and (iii) 10 Gy irradiation on a 0.35 T MR-Linac with real-time cine-MRI acquisition (IRrtMRI). Organoid viability was assessed by CellTiter-Glo luminescence assay. Morphometric analysis was performed using Bright-field images classifying organoids as small, medium, or large. Cell death was quantified by 7-AAD flow cytometry, while DNA damage and apoptosis were evaluated via western blotting for γH2AX and cleaved-PARP1. Statistical analysis was performed by ANOVA with multiple comparisons (GraphPad Prism 10). Results Both PDO lines exhibited reduced growth and viability after irradiation, with a significantly stronger effect under combined IRrtMRI exposure. The proportion of large organoids decreased to 0%, while small organoids markedly increased in both PDAC-9 and PDAC-12 (p < 0.01). Cell viability assays confirmed enhanced inhibition following IRrtMRI compared to IR alone (p < 0.001). Flow cytometry revealed that apoptotic cells increased from 40% (IR) to 70% (IRrtMRI) in PDAC-12, whereas PDAC-9 showed relative resistance. Western blot analysis demonstrated increased γH2AX phosphorylation in both PDOs and cleaved-PARP1 induction selectively in PDAC-12 under combined treatment, indicating augmented DNA damage and apoptosis. Conclusion Exposure to radiotherapy under a static 0.35 T magnetic field with real-time MRI enhances genotoxic and apoptotic effects in PDAC organoids compared to conventional irradiation. The observed radiosensitization may derive from magnetic field–mediated prolongation of radical lifetimes, absence of CBCT X-ray–induced adaptive responses, and DNA polarization phenomena. These findings suggest a possible biological advantage of MRIgRT beyond improved targeting accuracy, supporting further investigation into its translational and clinical implications in pancreatic cancer. Biological sciences/Cancer Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Pancreatic cancer remains one of the deadliest malignancies, accounting for approximately 25% of cancer-related deaths worldwide. Despite significant advances in diagnostic techniques and the development of promising multimodal treatment strategies, more than 80% of patients are diagnosed with unresectable or metastatic disease [ 1 – 3 ]. Locally advanced pancreatic cancer (LAPC) has several definitions but essentially is a non-metastasized pancreatic cancer, in which upfront resection is considered not beneficial due to extensive vascular involvement and consequent high chance of a nonradical resection. The current standard of care for LAPC typically involves systemic chemotherapy with either neoadjuvant or exclusive intent, followed by surgical resection or loco-regional therapies such as radiotherapy. In recent years, technological progress in radiotherapy has led to the introduction of advanced irradiation modalities, including Intensity Modulated Radiotherapy (IMRT), Volumetric Modulated Arc Therapy (VMAT), and Stereotactic Body Radiotherapy (SBRT), which have demonstrated improved local control and survival outcomes [ 4 ]. Within this evolving therapeutic landscape, image-guided radiotherapy (IGRT) has been used with the aim to minimise uncertainties in treatment delivery through the use of different forms of medical imaging at frequent intervals throughout the patient’s treatment course [ 5 ]. One recent IGRT development has been the integration of MRI with a radiotherapy treatment machine (Magnetic Resonance-guided Radiotherapy (MRIgRT), which has emerged as an innovative treatment modality [ 6 ]. Preliminary data suggest its potential in enhancing treatment response and reducing toxicity across several anatomical sites, with pancreatic cancer showing particularly encouraging results [ 7 , 8 ]. MRIgRT offers significant clinical advantages, including superior soft tissue contrast through on-board MRI imaging and the ability to perform online adaptive radiotherapy. This enables daily treatment plan modifications that optimize dose distribution and allow for safe dose escalation, potentially enhancing therapeutic efficacy [ 9 ]. Since 2014, two commercial MRIgRT systems have become available: the MRIdian system (ViewRay Technologies., USA), which integrates a 0.35 T MRI scanner with a 6 MV linear accelerator, and the Unity system (Elekta, Sweden), which combines a 1.5 T MRI scanner with a 7 MV linac [ 10 , 11 ]. Despite promising clinical outcomes, limited data exists regarding the biological interactions between photon irradiation and the static magnetic field (SMF) inherent to MRIgRT, particularly concerning potential synergistic effects on cell lethality and tissue damage. A few studies have examined the interaction between SMFs and radiation. These studies revealed cellular physiological changes and an altered yield or complexity of DNA damage [ 12 – 14 ]. Despite this limited knowledge about the interaction between SMFs and ionizing radiation, there is a common belief that synergistic effects could be stimulated. These include a modified DNA damage response, stabilization of reactive oxygen species (ROS), and altered intercellular signaling. As clinical data alone are insufficient to elucidate these mechanisms, human organoid models have emerged as a valuable tool for studying radiobiological mechanisms [ 15 – 17 ]. Organoids are three-dimensional primary cultures that can be reliably established and maintained in vitro . Critically, they preserve the morphological and genomic characteristics of the originating tissue and have been shown to accurately replicate responses to oncological treatments and computational modeling [ 18 ]. Given the known interactions between biological systems and SMF, such as electrodynamic, magneto-mechanical, and electron spin effects—it is plausible that SMF exposure could modulate tissue responses to radiation, potentially enhancing the therapeutic effect. However, the literature on SMF-radiation interactions remains scarce and often outdated [ 12 ]. Notably, no studies to date have investigated the effects of irradiation in combination with a modern 0.35 T hybrid MR-linac on organoid models. Only a single, anecdotal study combining radiotherapy with a 1.5 T hybrid unit has been reported, yielding inconclusive results [ 19 ]. These discrepancies underscore a critical gap in our understanding of the biological mechanisms underpinning radiation-induced damage of the tumor in the presence of SMF. MATERIAL AND METHODS Patient-derived organoids culture Patient-derived pancreatic organoids were established from biopsy as previously described by Ruta et al. [ 20 ], with minor modifications. Tumor tissues were placed in 60 mm Petri dishes containing AdDF+++ culture medium (Advanced DMEM/F12 containing 1×Glutamax, 10 mM HEPES and antibiotics). Part of the tissue was fixed in formalin for histopathological and immunohistochemistry analysis; part was stored at − 80°C for DNA/RNA isolation. The remaining part was minced by surgical blades into small fragments for organoids generation and digested in 10 mL AdDF+++ supplemented with 5 µM RHO/ROCK pathway inhibitor (Y-27632, Tocris) containing 2 mg/mL Collagenase II (Thermo Fisher Scientific) DispaseII and DNAse I (Sigma-Aldrich) on an orbital shaker at 37°C for one hour. The cell suspension was then applied to a MACS SmartStrainer (70 µm), placed on a 50 mL tube and washed with 10 mL of AdDF+ + +culture medium and centrifugated at 290 g. The pellet was incubated with 1 mL red blood cell lysis buffer for 5 minutes (min) at room temperature to eliminate erythrocytes, followed by washing with culture medium and pelleting at 290 g. Cells were embedded in undiluted (100%) Cultrex growth factor reduced BME type 2 (Bio-Techne) on ice and 40 µL drops of BME cell suspension were allowed to solidify to a pre-warmed 24 well suspension cell plates (Greiner). The plate was placed at 37°C for 30 min to allow the Matrigel to polymerize before being overlaid with 500 µL of a growth factor cocktail medium [AdDF+ + +culture medium supplemented with EGF (50 ng/mL Peprotech), A83-01 (0.5 µM), B27 (1X GIBCO) and Primocin (1 mg/mL, InvivoGen), Y-27632 (9 µM Tocris), Wnt3a-conditioned medium (50% v/v), recombinant R-Spondin 1 (RSPO1)-conditioned medium (10% v/v) Nicotinamide (10 mM, Sigma-Aldrich), Gastrin (10 nM, Biogems), fibroblast growth factor 10 (FGF10, 100 ng/mL, Peprotech), N-acetyl-L-cysteine (1 mM, Sigma-Aldrich) and Noggin (0.1 mg/mL, Peprotech)] and incubated at 37°C in humidified air containing 5% CO2. Medium was changed every 3–4 days and organoids were passaged every 1–2 weeks at suitable ratio (1:1 to 1:3) . Treatment conditions Two PDCA patient-derived pancreatic organoid lines were treated following a previously established protocol: each organoid line was plated in four separate 24-well plates, corresponding to four different treatment conditions: 1. Control (CTRL): Organoids were not exposed to radiations. 2. Irradiation on a standard linear accelerator (IR): Organoids were irradiated using a conventional linear accelerator (TrueBeam Edge, Varian) delivering 6FFF MV photon beams at a dose rate of 600 MU/min. A single dose of 10 Gy was delivered using a rectangular anterior–posterior field, positioning the samples at the isocenter. This dose level was selected to simulate a standard single-fraction stereotactic body radiation therapy (SBRT) session as employed in clinical practice. The experimental setup is shown in Fig. 1 A. 3. Irradiation in MRI-LINAC with real-time MRI guidance (IRrtMRI): irradiation was performed with the MRIdian system at 0.35 T and a single dose of 10 Gy, maintaining the same field configuration. Additionally, real-time cine-MRI was acquired during irradiation using a TrueFISP (true fast imaging with steady-state free precession) sequence at 8 frames per second in a sagittal plane. Experimental setup is shown in Fig. 1 B. Viability assays Dissociated Patient-Derived Organoids (PDOs) were resuspended in 2% BME/growth medium and seeded in 100 µl volume on BME pre-coated 96-well plates after 72 hours. After seven days the viability was determined by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) using a microplate reader (Spark, Tecan). Viability was determined relative to the untreated control cells and analysed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). Organoid size measurement and classification The size of organoids was measured as described in Morrow et al [ 21 ]. A straight line was drawn across the widest part for each organoid to determine its diameter using ImageJ software. This process was repeated until the diameter of each organoid in the sample was measured. To classify organoids by size, the diameter range was first calculated by subtracting the diameter of the smallest organoid from that of the largest, then divided by three. The result determined the range to define small, medium and large organoid categories. Each organoid was subsequently assigned to one of these size groups based on its measured diameter. All measurements were performed in triplicate to ensure reproducibility. FACS analysis PDO cells were fixed and stored in 70% ethanol for up to one week. After washing with DPBS, 10 6 cells were stained with 0.3 mL of 10 µg/ml 7-AAD (7-amino-actinomycin D) in DPBS supplemented with protease inhibitors and RNAsin (1:1000). After 30 min of incubation at 4°C in the dark, cells were sorted by DNA content using DxFLEX Beckman Coulter. Western blot analysis Whole cell proteins were obtained by lysing the organoids with RIPA lysis buffer (60mM Tris-HCl pH 7.5, 1% NP40, 150mM NaCl, 50mM NaF, 2mM EDTA) supplemented with 2mM Na- orthovanadate, 1mM DTT and protease-inhibitors. Lysates were incubated on ice for 30 min, briefly sonicated, and centrifuged (15 min at 15 000 rpm, 4°C). Protein extracts were quantified by Bradford assays, diluted in SDS-sample buffer and boiled at 95° for five min. Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis, blotted to PVDF (Polyvinylidene fluoride), and transferred overnight. After blocking in 5% non-fat milk, membranes were probed with the following primary antibodies: 1:1000 anti- γH2AX (Cell Signalling), 1:1000 anti-PARP (Cell Signalling) and 1:1000 anti– βTubulin (Cell Signalling), at 4°C, overnight. After incubation with secondary horseradish peroxidase-conjugated antibody, 1:5000 dilution for one hour, proteins were visualized by the enhanced chemiluminescence system (Bio-Rad or Amersham Biosciences, Buckinghamshire, UK) using a ChemiDoc imaging system (UVITEC, Cambridge). Statistical analysis Cell proliferation, apoptosis and western blot data were analysed by one-way analysis of variance (ANOVA), using Tukey’s multiple comparison test to determine if significant differences existed between groups. PDO size data were evaluated by the two-way analysis of variance (ANOVA), using Bonferroni’s multiple comparisons test. All data were reported as mean ± SD (n ≥ 3) and statistical analyses were performed using the GraphPad Prism10 Software. A p-value ≤ 0.05 was considered significant. Ethics statement Human pancreatic ductal adenocarcinoma (PDAC) tissues used for the generation of patient-derived organoids were obtained from treatment-naïve patients undergoing biopsy at Fondazione Policlinico Universitario “A. Gemelli” IRCCS (Rome, Italy). All experimental procedures involving human biological samples were performed in accordance with relevant guidelines and regulations and with the Declaration of Helsinki. The study protocol was approved by the Institutional Ethics Committee of Fondazione Policlinico Universitario “A. Gemelli” IRCCS. Written informed consent was obtained from all patients prior to sample collection. RESULTS Two PDAC PDOs, PDAC-9 and PDAC-12, were established from treatment-naïve patients and preserved genetic phenotypes of the corresponding tissues, as previously reported [ 20 ]. To evaluate the impact of irradiation in combination with real-time cine-MRI on PDAC organoid growth ex vivo , the study comprised three experimental groups (see M&M for details): 1. Control (CTRL): PDO were not exposed to radiations. 2. Irradiation of PDO on a standard linear accelerator (IR): experimental setup shown in Fig. 1 A. 3. Irradiation in MRI-LINAC with real-time MRI guidance (IRrtMRI): experimental setup is shown in Fig. 1 B. To directly assess the impact of treatments on PDAC organoid growth ex vivo , PDOs were γ-irradiated with 10 Gy and analysed after 72 h from irradiation as illustrated in the scheme of the study workflow (Fig. 2 A). Morphological evaluation of the PDAC-9 and PDAC-12 PDOs, grown in 3D cultures for 13 days after treatments, indicated that both irradiations alone and its IRrtMRI combination caused a reduction of proliferation as shown in representative brightfield images in Fig. 2 B and 2 C. To directly assess the impact of treatments on the self-renewal ability of PDOs, we passaged them after 72 h from irradiation and measured the size of the newly formed organoids after additional seven days. As illustrated in Fig. 2 C, PDAC-9 and PDAC-12 exhibited a significant reduction in the number and size of the organoids formed after treatment, showing a higher impact of the IRrtMRI combination exposure. A quantitative evaluation of this effect was carried out by measuring the size of the organoids, classified in three groups small, medium and large. For both PDAC-9 and PDAC-12 organoids, the percentage of the “large size” subgroup dropped to zero after both IR and IRrtMRI treatments, while the percentage of “small size” PDOs increased concomitantly with greater effect in IRrtMRI combination treatment. For the “medium size” subgroup, the greater inhibitory effect was shown after IRrtMRI treatment for both PDOs (Fig. 2 C). Importantly, the result of the morphological analysis was in line with the data of the viability assay, which showed that the combined IRrtMRI treatment significantly enhanced the inhibitory effect of IR on organoid cell viability in both PDO lines (Fig. 2 D). To determine if the inhibition of cell viability is due to reduced growth of PDOs accompanied by cell death, we also performed 7-AAD staining to assess the DNA content in PDOs at 72h after irradiation. Flow cytometry analysis after 7-AAD staining showed that 40% of the cells of the PDAC-12 PDO were apoptotic (sub-G1 phase DNA content) and this effect was significantly increased to 70% in PDOs exposed to the IRrtMRI combination (Fig. 3 A). In contrast, no significant effect of these treatments on cell death was observed in PDAC-9. These results were confirmed by western blot analysis of cleaved-PARP1 protein, a marker of apoptosis, which was only induced in PDAC-12 cells after IRrtMRI combined treatment (Fig. 3 B). On the other hand, the phosphorylation of γH2AX, a marker of DNA damage, was significantly increased in both PDO lines exposed to the combined IRrtMRI treatment compared to IR alone (Fig. 3 B). In conclusion, these data indicate that concurrent exposure to IR and rtMRI exerts a stronger antiproliferative effect on PDAC PDOs in comparison to IR treatment alone, suggesting that the combination treatment enhances the inhibitory effects of IR in PDAC PDOs. Discussion The results obtained on two pancreatic ductal adenocarcinoma PDOs, PDAC-9 and PDCA-12, indicate that irradiation in the presence of a SMF, combined with real-time MRI acquisition, induces a stronger inhibition of cell growth than irradiation alone, intensifying genotoxic damage and promoting apoptosis, as demonstrated by the increased phosphorylation of γH2AX and the induction of cleaved PARP. Four mechanisms described in the literature provide a useful interpretative framework for these findings. Firstly, static magnetic fields can prolong the lifetime of free radicals in the triplet state, thereby extending the time window for interactions with DNA and amplifying radiation-induced damage. This mechanism, reported by the UCLA group [ 22 ], has been associated with increased cellular lethality both in vitro and in vivo under weak magnetic fields. In this context, the enhanced γH2AX phosphorylation and apoptotic response observed in our experiments are consistent with the hypothesis that the static magnetic field acts as a physical co-factor capable of potentiating the effects of radiotherapy. Secondly, the opposite effect has been reported in X-ray–guided radiotherapy systems, where low-dose imaging can trigger an adaptive response mediated by survivin, leading to tumor radioprotection. This phenomenon, described by the Chicago group [ 23 ], suggests that imaging modality can influence tumor biology independently of the therapeutic irradiation. In our model, the use of MRIgRT eliminates X-ray exposure during imaging and thus reduces the likelihood of inducing such a radioprotective response, providing an additional biological advantage that complements the potential radiosensitizing effect of the magnetic field. Thirdly, static magnetic fields have been reported to induce partial orientation or polarization of DNA due to the diamagnetic anisotropy of nucleic acid bases. This phenomenon, first described by Maret et al. [ 24 ] and later elaborated by Vavrinská et al. [ 25 ], can increase the surface area of DNA exposed to radiation damage while simultaneously interfering with the accessibility of DNA repair proteins, thereby delaying the resolution of double-strand breaks. In such context, in PDCA-9 and PDCA-12 the observed increased phosphorylation of γH2AX could be the result of both initial DNA damage and impaired or delayed DNA repair. Fourth, Wishart et al. demonstrated that the presence of a moderate-intensity static magnetic field (1.5 T) significantly enhanced X-ray-induced apoptosis in a three-dimensional pancreatic cancer model, with effects particularly pronounced under hypoxic conditions that closely resemble the pancreatic tumor microenvironment [ 16 ]. These results suggest that the magnetic field intrinsic to MRIgRT systems may contribute to biological radiosensitization beyond physical dose delivery. In addition, earlier in vitro studies summarized by Dini and Abbro reported that static magnetic fields of moderate intensity can modulate key cellular processes, including proliferation, apoptosis, ion fluxes, and oxidative stress responses, in a cell-type- and context-dependent manner [ 17 ]. Despite these possible explanations, it is important to note that the existing literature reports conflicting results. Studies conducted at 1 Tesla [ 20 ] or using the Unity platform did not find significant differences in biological response to the combination of radiotherapy and SMF, whereas other experimental observations reported radiosensitizing effects attributable to the magnetic field [ 21 ]. Some preliminary reports have even suggested that sample heating during MRI acquisition may act as a confounding factor. Our investigation, based on the use of PDOs, represents an innovative approach compared with conventional experimental radiobiological models. PDOs are three-dimensional structures generated directly from patient tumor samples that faithfully recapitulate the histological architecture of the original tissue. This includes the spatial organization of cells, cell–cell and cell–matrix interactions, as well as gradients of nutrients, oxygen, and growth factors. Unlike traditional two-dimensional (2D) culture systems, in which homogeneous cell lines are grown on flat artificial surfaces, PDOs preserve the phenotypic heterogeneity of the primary tumor, providing a more accurate reflection of intratumoral diversity and therapeutic response. This model is particularly effective for the generation of experimental data that are physiologically more relevant and predictive, thereby improving the translational potential of preclinical studies and their applicability to clinical settings [ 15 – 17 ]. In this context, our study provides an original contribution by exploiting the unique features of PDOs to investigate biological mechanisms and treatment responses in a pre-clinical model that more closely mirrors patient tumor biology than conventional 2D cultures. Conclusions This study demonstrates that the combination of irradiation, static magnetic fields, and real-time MRI acquisition intensifies DNA damage and apoptotic activation in pancreatic cancer–derived organoids. Our findings can be interpreted in light of three mechanisms: the prolonged lifetime of free radicals in the presence of magnetic fields, which amplifies genotoxic damage [ 22 ]; the absence of X-ray positioning imaging acquired seconds to minutes before treatment fraction delivery, which eliminates the possibility of a survivin-mediated adaptive radioprotective response [ 23 ]; and magnetic field–induced DNA polarization, which may increase DNA exposure to damage and interfere with repair processes [ 24 , 25 ]. This tripartite interpretative framework suggests that MRIgRT may not only offer technical advantages in unprecedented target volume visualization and online adaptation, but it could also confer an intrinsic biological benefit compared with X-ray–guided radiotherapy. Our work provides a robust proof of principle supporting the value of PDOs as a platform for investigating innovative combinations of magnetic fields and radiotherapy. However, the different responses observed among two PDO lines, probably due to the intrinsic genetic and molecular features or interpatient heterogeneity, underscores the need for further studies in a larger cohort of PDOs to identify the most responsive patient subgroups and the specific contributions of radical-mediated effects, DNA polarization, imaging-related mechanisms, and potential confounding factors such as MRI-induced heating. Moreover, further research is required to understand the specific mechanisms underlying the stronger antiproliferative effect of IRrtMRI combination exposure and demonstrate its selective effect on pancreatic tumor cells in comparison to normal cells. Overall, the data presented here strengthens the biological rationale for the development and clinical validation of MR-linac based therapeutic protocols, potentially improving radiotherapeutic outcomes for pancreatic cancer. Declarations Data Availability The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements The authors acknowledge the support of Prof. Michael Chuong from Miami Cancer Institute, Miami, Florida (USA) in reviewing this paper. Research funding The authors declare support of Radius srl of Budrio (BO), Italy, for funding these experiments. Author Contributions Statement L.B. and C.G.C. conceived and designed the study and supervised the overall research project. L.B., C.G.C., L.P., G.C., M.N., M.G., A.R., and G.P. contributed to radiotherapy planning, experimental irradiation procedures, data acquisition, and interpretation of radiobiological results. A.C., C.C., E.C., and R.V. established and maintained the patient-derived organoid models and performed organoid-based experiments and analyses. R.V. and C.S. contributed to the biological interpretation of the data and to the analysis of molecular and cellular endpoints. K.B. provided critical expertise in radiobiology and contributed to data interpretation and manuscript revision. R.M., V.T., S.A., and G.T. contributed to patient recruitment, clinical characterization, and provided clinical and translational input relevant to pancreatic cancer. L.B., C.G.C., and M.A.G. drafted the manuscript. All authors contributed to critical revision of the manuscript for important intellectual content. All authors approved the final version of the manuscript and agree to be accountable for their own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. References Capurso, G. & Sette, C. Drug resistance in pancreatic cancer: New player caught in act. EBioMedicine 40 , 39–40. https://doi.org/10.1016/j.ebiom.2019.02.008 (2019). Hidalgo, M. Pancreatic cancer. N Engl. J. Med. 362 , 1605–1617. https://doi.org/10.1056/NEJMra0901557 (2010). Mohammed, S., Van Buren, G. & Fisher, W. E. Pancreatic cancer: advances in treatment. World J. Gastroenterol. 20 , 9354–9360. https://doi.org/10.3748/wjg.v20.i28.9354 (2014). Lominska, C. E., Unger, K., Nasr, N. M., Haddad, N. & Gagnon, G. Stereotactic body radiation therapy for reirradiation of localized adenocarcinoma of the pancreas. Radiat. Oncol. 7 , 74. https://doi.org/10.1186/1748-717X-7-74 (2012). Franzone, P. et al. Image-guided radiation therapy (IGRT): practical recommendations of Italian Association of Radiation Oncology (AIRO). Radiol. Med. 121 , 958–965. https://doi.org/10.1007/s11547-016-0674-x (2016). Chin, S. et al. Magnetic resonance-guided radiation therapy: A review. J. Med. Imaging Radiat. Oncol. 64 , 163–177. https://doi.org/10.1111/1754-9485.12968 (2020). Boldrini, L. et al. Online adaptive magnetic resonance guided radiotherapy for pancreatic cancer: state of the art, pearls and pitfalls. Radiat. Oncol. 14 , 71. https://doi.org/10.1186/s13014-019-1275-3 (2019). Placidi, L. et al. On-line adaptive MR guided radiotherapy for locally advanced pancreatic cancer: Clinical and dosimetric considerations. Tech. Innov. Patient Support Radiat. Oncol. 15 , 15–21. https://doi.org/10.1016/j.tipsro.2020.06.001 (2020). Corradini, S. et al. MR-guidance in clinical reality: current treatment challenges and future perspectives. Radiat. Oncol. 14 , 92. https://doi.org/10.1186/s13014-019-1308-y (2019). Klüter, S. Technical design and concept of a 0.35 T MR-Linac. Clin. Transl Radiat. Oncol. 18 , 98–101. https://doi.org/10.1016/j.ctro.2019.04.007 (2019). Raaymakers, B. W. et al. Integrating a 1.5 T MRI scanner with a 6 MV accelerator: proof of concept. Phys. Med. Biol. 54 , N229–237. https://doi.org/10.1088/0031-9155/54/12/N01 (2009). Mohajer, J. K., Nisbet, A., Velliou, E., Ajaz, M. & Schettino, G. Biological effects of static magnetic field exposure in the context of MR-guided radiotherapy. Br. J. Radiol. 92 , 20180484. https://doi.org/10.1259/bjr.20180484 (2019). Wishart, G., Gupta, P., Nisbet, A., Velliou, E. & Schettino, G. Enhanced effect of X-rays in the presence of a static magnetic field within a 3D pancreatic cancer model. Br. J. Radiol. 96 (1143), 20220832. 10.1259/bjr.20220832 (2023). Epub 2023 Jan 14. PMID: 36475863; PMCID: PMC9975369. Dini, L. & Abbro, L. Bioeffects of moderate-intensity static magnetic fields on cell cultures. Micron 36 (3), 195–217. 10.1016/j.micron.2004.12.009 (2005). Epub 2005 Jan 25. PMID: 15725590. Fang, Z., Li, P., Du, F., Shang, L. & Li, L. The role of organoids in cancer research. Exp. Hematol. Oncol. 12 , 69. https://doi.org/10.1186/s40164-023-00433-y (2023). Eke, I. & Cordes, N. Radiobiology goes 3D: how ECM and cell morphology impact on cell survival after irradiation. Radiother Oncol. 99 , 271–278. https://doi.org/10.1016/j.radonc.2011.06.007 (2011). Yu, J., Wang, K., Tang, Y. & Zheng, D. Applications and perspectives of tumor organoids in radiobiology (Review). Oncol. Rep. 52 , 100. https://doi.org/10.3892/or.2024.8759 (2024). Driehuis, E. et al. Pancreatic cancer organoids recapitulate disease and allow personalized drug screening. Proc. Natl. Acad. Sci. U S A . 116 , 26580–26590. 10.1073/pnas.1911273116 (2019). Nicosia, L. et al. Combinatorial Effect of Magnetic Field and Radiotherapy in PDAC Organoids: A Pilot Study. Biomedicines 8 , 609. https://doi.org/10.3390/biomedicines8120609 (2020). Ruta, V. et al. An alternative splicing signature defines the basal-like phenotype and predicts worse clinical outcome in pancreatic cancer. Cell. Rep. Med. 5 (2), 101411. 10.1016/j.xcrm.2024.101411 (2024). Epub 2024 Feb 6. PMID: 38325381; PMCID: PMC10897606. Morrow, R. J., Ernst, M. & Poh, A. R. Longitudinal quantification of mouse gastric tumor organoid viability and growth using luminescence and microscopy. STAR. Protoc. 4 , 102110. https://doi.org/10.1016/j.xpro.2023.102110 (2023). Iwamoto, K. S. et al. Weak Magnetic Fields Enhance the Efficacy of Radiation Therapy. Adv. Radiat. Oncol. 6 , 100645. https://doi.org/10.1016/j.adro.2021.100645 (2021). Grdina, D. J. et al. A survivin-associated adaptive response in radiation therapy. Cancer Res. 73 , 4418–4428. https://doi.org/10.1158/0008-5472.CAN-12-4640 (2013). Maret, G. Orientation of Nucleic Acids in High Magnetic Fields. Phys. Rev. Lett. 35 , 397–400. https://doi.org/10.1103/PhysRevLett.35.397 (1975). Vavrinská, A. et al. Impact of nucleic acid self-alignment in a strong magnetic field on the interpretation of indirect spin-spin interactions. J. Biomol. NMR . 64 , 53–62. https://doi.org/10.1007/s10858-015-0005-x (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9009316","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":607575990,"identity":"690ea8d0-d910-4649-ba05-c051564a8af0","order_by":0,"name":"Luca Boldrini","email":"","orcid":"","institution":"Fondazione Policlinico Universitario “A. Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Boldrini","suffix":""},{"id":607575991,"identity":"ed9e1922-d099-4cab-a9e7-0bbc82ed1364","order_by":1,"name":"Carlo Gugliemo Cattaneo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYHACxgNIHBuQQOMB7CoRAKyAhw3MTgNpaSBJy2GECC7A3372wYEPv+zk7eWbnz34UHHebm37YaAtNTbRuLRInEk3ODizL9mwh43N3HDGmdvJ284kArUcS8ttwKHFAOj6w7w9Bxh72BjMpHnbbiebHQBqYWw4jFsL/zOwFvseNvZv0n/bziWbnX9IQIsE0BaeHwcSe9h4zKQZ2w7Ymd0gYIvEjWcMB2c2JCf3HMspk+w5k5xgdgNoSwIev/D3pzE++PDHzra9+fg2iR8VdvZm59MfPvhQY4NTCxgwtiHYiWCVCfiUg8EfBNOeoOJRMApGwSgYcQAAQGJlBKUavrAAAAAASUVORK5CYII=","orcid":"","institution":"Fondazione Policlinico Universitario “A. 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Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Corti","middleName":"","lastName":"Christian","suffix":""},{"id":607575995,"identity":"eb578375-f812-470c-a410-1c6bd086d9f3","order_by":5,"name":"Eleonora Cesari","email":"","orcid":"","institution":"GSTeP Organoids Research Core Facility, Fondazione Policlinico Universitario “A. 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Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Giuditta","middleName":"","lastName":"Chiloiro","suffix":""},{"id":607575999,"identity":"dd4f1c61-5336-4baf-b6f1-593900f55587","order_by":9,"name":"Matteo Nardini","email":"","orcid":"","institution":"Fondazione Policlinico Universitario “A. Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Matteo","middleName":"","lastName":"Nardini","suffix":""},{"id":607576000,"identity":"d490b8a1-2bfc-4b8d-a09a-79315d770875","order_by":10,"name":"Matteo Galetto","email":"","orcid":"","institution":"Fondazione Policlinico Universitario “A. Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Matteo","middleName":"","lastName":"Galetto","suffix":""},{"id":607576001,"identity":"34848f95-a2c6-47e9-ba39-4b8f7ae36954","order_by":11,"name":"Angela Romano","email":"","orcid":"","institution":"Fondazione Policlinico Universitario “A. Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Angela","middleName":"","lastName":"Romano","suffix":""},{"id":607576002,"identity":"c2a230cb-2310-4ca5-bc6a-b7be541c1a6c","order_by":12,"name":"Giulia Panza","email":"","orcid":"","institution":"Fondazione Policlinico Universitario “A. Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"","lastName":"Panza","suffix":""},{"id":607576003,"identity":"e4a4e33e-4755-4c60-a9d8-e620cc398005","order_by":13,"name":"Roberta Menghi","email":"","orcid":"","institution":"Gemelli Pancreatic Center, Fondazione Policlinico Universitario \"Agostino Gemelli\" IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Menghi","suffix":""},{"id":607576004,"identity":"834dd42c-a948-4e47-9b45-bf6b69becbb7","order_by":14,"name":"Vincenzo Tondolo","email":"","orcid":"","institution":"Department of translational Medicine and Surgery, Università Cattolica del Sacro Cuore","correspondingAuthor":false,"prefix":"","firstName":"Vincenzo","middleName":"","lastName":"Tondolo","suffix":""},{"id":607576005,"identity":"908d2efd-6a8f-4f13-b654-280a203e5a3c","order_by":15,"name":"Sergio Alfieri","email":"","orcid":"","institution":"Department of translational Medicine and Surgery, Università Cattolica del Sacro Cuore","correspondingAuthor":false,"prefix":"","firstName":"Sergio","middleName":"","lastName":"Alfieri","suffix":""},{"id":607576006,"identity":"7d1e9e32-7cc0-4a4f-bba6-5d3d6399d05d","order_by":16,"name":"Giampaolo Tortora","email":"","orcid":"","institution":"Department of translational Medicine and Surgery, Università Cattolica del Sacro Cuore","correspondingAuthor":false,"prefix":"","firstName":"Giampaolo","middleName":"","lastName":"Tortora","suffix":""},{"id":607576007,"identity":"ce2ed910-cfa3-4e08-8566-e46adf165e91","order_by":17,"name":"Claudio Sette","email":"","orcid":"","institution":"Department of Neuroscience, Section of Human Anatomy, Università Cattolica del Sacro Cuoreit","correspondingAuthor":false,"prefix":"","firstName":"Claudio","middleName":"","lastName":"Sette","suffix":""},{"id":607576008,"identity":"18e46aba-a8d8-4e00-a17a-59a9e2bb143e","order_by":18,"name":"Maria Antonietta Gambacorta","email":"","orcid":"","institution":"Fondazione Policlinico Universitario “A. Gemelli” IRCCS","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Antonietta","lastName":"Gambacorta","suffix":""}],"badges":[],"createdAt":"2026-03-02 11:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9009316/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9009316/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104998249,"identity":"80f1512c-4e89-462b-92bc-5c80d9c6098f","added_by":"auto","created_at":"2026-03-19 16:25:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":732296,"visible":true,"origin":"","legend":"\u003cp\u003eA-B. Schematic representation of the irradiation setup used for PDO experiments. The upper panels illustrate the beam geometry and irradiation field, while the lower panels show the corresponding top views of sample positioning. The PDO-containing region is centered within the field to ensure uniform dose delivery.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9009316/v1/9b5833a68bd952f3c2167cde.png"},{"id":104998341,"identity":"a129bff5-98ba-416a-b73a-766d50047a0f","added_by":"auto","created_at":"2026-03-19 16:26:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":911397,"visible":true,"origin":"","legend":"\u003cp\u003eResponse to irradiation and IRrtMRI of PDO. \u003cstrong\u003eA\u003c/strong\u003e) Scheme of the study workflow. At 72h after irradiation, PDOs were subjected to i) protein extraction for WB analysis; ii) cell dissociation and seeding in 96-well plates to assess self-renewal ability of organoids and viability using CellTiter-Glo luminescent assay after 7 days; iii) cell dissociation and plating in BME droplets to assess the growth. \u003cstrong\u003eB\u003c/strong\u003e) Representative brightfield images of PDAC PDO cultures after 10 days in BME droplets. Scale bar: 500 μm; \u003cstrong\u003eC\u003c/strong\u003e) Bright-field images of PDO cultures irradiated alone or in combination with IRrtMRI (left panel) (Scale bar: 200 μm). Percentage of large, medium, and small PDOs for conditions (mean ± SD, n = 3) (∗: p ≤ 0.05, ∗∗: p ≤ 0.01, ∗∗∗: p ≤ 0.001; ∗∗∗∗: p ≤ 0.0001) (right panel). \u003cstrong\u003eD\u003c/strong\u003e) After 72h after irradiation PDO were dissociated and seeded in 96 well plate. After 7 days, living cells were measured using Cell Titer Glo 3D assay (mean ± D, n = 12) (∗∗∗: p ≤ 0.001; ∗∗∗∗: p ≤ 0.0001).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9009316/v1/a5543f50f1f05afb232dc1b2.png"},{"id":104998284,"identity":"c5bf1d14-94bf-4634-8611-4e69f15cb15f","added_by":"auto","created_at":"2026-03-19 16:26:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":631449,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of irradiation on PDOs viability. \u003cstrong\u003eA\u003c/strong\u003e) Flow cytometry analysis of apoptotic cells associated with sub-G1 DNA content. Representative histograms of cell cycle distribution depicting apoptosis in PDO cells irradiated alone or in combination with IRrtMRI. The percent of sub-G1 is measured as the percentage of the number of cells in the sub-G1 population relative to the number of total cells. Quantification of sub-G1 phase was analyzed by CyExpert software (mean±SD) \u003cstrong\u003eB\u003c/strong\u003e) Western blot and densitometric analyses using β-tubulin as loading control (mean ± SD, n = 3, one-way analysis of variance ANOVA, using Tukey's multiple comparison test; ∗: p ≤ 0.05).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9009316/v1/4bf81a2f22d8c69883816dc6.png"},{"id":106384837,"identity":"23ec6211-6e3f-41ee-ba98-9b0aeacff46b","added_by":"auto","created_at":"2026-04-08 05:57:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2999166,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9009316/v1/3e4d3a51-bac6-4cc9-b799-08110c7ea9fb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased cytotoxic effects via combined treatment of magnetic field and radiation therapy in patient-derived organoids of pancreatic ductal adenocarcinoma","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePancreatic cancer remains one of the deadliest malignancies, accounting for approximately 25% of cancer-related deaths worldwide. Despite significant advances in diagnostic techniques and the development of promising multimodal treatment strategies, more than 80% of patients are diagnosed with unresectable or metastatic disease [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Locally advanced pancreatic cancer (LAPC) has several definitions but essentially is a non-metastasized pancreatic cancer, in which upfront resection is considered not beneficial due to extensive vascular involvement and consequent high chance of a nonradical resection. The current standard of care for LAPC typically involves systemic chemotherapy with either neoadjuvant or exclusive intent, followed by surgical resection or loco-regional therapies such as radiotherapy.\u003c/p\u003e \u003cp\u003eIn recent years, technological progress in radiotherapy has led to the introduction of advanced irradiation modalities, including Intensity Modulated Radiotherapy (IMRT), Volumetric Modulated Arc Therapy (VMAT), and Stereotactic Body Radiotherapy (SBRT), which have demonstrated improved local control and survival outcomes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Within this evolving therapeutic landscape, image-guided radiotherapy (IGRT) has been used with the aim to minimise uncertainties in treatment delivery through the use of different forms of medical imaging at frequent intervals throughout the patient\u0026rsquo;s treatment course [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne recent IGRT development has been the integration of MRI with a radiotherapy treatment machine (Magnetic Resonance-guided Radiotherapy (MRIgRT), which has emerged as an innovative treatment modality [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Preliminary data suggest its potential in enhancing treatment response and reducing toxicity across several anatomical sites, with pancreatic cancer showing particularly encouraging results [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMRIgRT offers significant clinical advantages, including superior soft tissue contrast through on-board MRI imaging and the ability to perform online adaptive radiotherapy. This enables daily treatment plan modifications that optimize dose distribution and allow for safe dose escalation, potentially enhancing therapeutic efficacy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Since 2014, two commercial MRIgRT systems have become available: the MRIdian system (ViewRay Technologies., USA), which integrates a 0.35 T MRI scanner with a 6 MV linear accelerator, and the Unity system (Elekta, Sweden), which combines a 1.5 T MRI scanner with a 7 MV linac [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite promising clinical outcomes, limited data exists regarding the biological interactions between photon irradiation and the static magnetic field (SMF) inherent to MRIgRT, particularly concerning potential synergistic effects on cell lethality and tissue damage. A few studies have examined the interaction between SMFs and radiation. These studies revealed cellular physiological changes and an altered yield or complexity of DNA damage [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Despite this limited knowledge about the interaction between SMFs and ionizing radiation, there is a common belief that synergistic effects could be stimulated. These include a modified DNA damage response, stabilization of reactive oxygen species (ROS), and altered intercellular signaling. As clinical data alone are insufficient to elucidate these mechanisms, human organoid models have emerged as a valuable tool for studying radiobiological mechanisms [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Organoids are three-dimensional primary cultures that can be reliably established and maintained \u003cem\u003ein vitro\u003c/em\u003e. Critically, they preserve the morphological and genomic characteristics of the originating tissue and have been shown to accurately replicate responses to oncological treatments and computational modeling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the known interactions between biological systems and SMF, such as electrodynamic, magneto-mechanical, and electron spin effects\u0026mdash;it is plausible that SMF exposure could modulate tissue responses to radiation, potentially enhancing the therapeutic effect. However, the literature on SMF-radiation interactions remains scarce and often outdated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Notably, no studies to date have investigated the effects of irradiation in combination with a modern 0.35 T hybrid MR-linac on organoid models. Only a single, anecdotal study combining radiotherapy with a 1.5 T hybrid unit has been reported, yielding inconclusive results [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese discrepancies underscore a critical gap in our understanding of the biological mechanisms underpinning radiation-induced damage of the tumor in the presence of SMF.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient-derived organoids culture\u003c/h2\u003e \u003cp\u003ePatient-derived pancreatic organoids were established from biopsy as previously described by Ruta et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], with minor modifications. Tumor tissues were placed in 60 mm Petri dishes containing AdDF+++ culture medium (Advanced DMEM/F12 containing 1\u0026times;Glutamax, 10 mM HEPES and antibiotics). Part of the tissue was fixed in formalin for histopathological and immunohistochemistry analysis; part was stored at \u0026minus;\u0026thinsp;80\u0026deg;C for DNA/RNA isolation. The remaining part was minced by surgical blades into small fragments for organoids generation and digested in 10 mL AdDF+++ supplemented with 5 \u0026micro;M RHO/ROCK pathway inhibitor (Y-27632, Tocris) containing 2 mg/mL Collagenase II (Thermo Fisher Scientific) DispaseII and DNAse I (Sigma-Aldrich) on an orbital shaker at 37\u0026deg;C for one hour. The cell suspension was then applied to a MACS SmartStrainer (70 \u0026micro;m), placed on a 50 mL tube and washed with 10 mL of AdDF+ + +culture medium and centrifugated at 290 g. The pellet was incubated with 1 mL red blood cell lysis buffer for 5 minutes (min) at room temperature to eliminate erythrocytes, followed by washing with culture medium and pelleting at 290 g. Cells were embedded in undiluted (100%) Cultrex growth factor reduced BME type 2 (Bio-Techne) on ice and 40 \u0026micro;L drops of BME cell suspension were allowed to solidify to a pre-warmed 24 well suspension cell plates (Greiner). The plate was placed at 37\u0026deg;C for 30 min to allow the Matrigel to polymerize before being overlaid with 500 \u0026micro;L of a growth factor cocktail medium [AdDF+ + +culture medium supplemented with EGF (50 ng/mL Peprotech), A83-01 (0.5 \u0026micro;M), B27 (1X GIBCO) and Primocin (1 mg/mL, InvivoGen), Y-27632 (9 \u0026micro;M Tocris), Wnt3a-conditioned medium (50% v/v), recombinant R-Spondin 1 (RSPO1)-conditioned medium (10% v/v) Nicotinamide (10 mM, Sigma-Aldrich), Gastrin (10 nM, Biogems), fibroblast growth factor 10 (FGF10, 100 ng/mL, Peprotech), N-acetyl-L-cysteine (1 mM, Sigma-Aldrich) and Noggin (0.1 mg/mL, Peprotech)] and incubated at 37\u0026deg;C in humidified air containing 5% CO2. Medium was changed every 3\u0026ndash;4 days and organoids were passaged every 1\u0026ndash;2 weeks at suitable ratio (1:1 to 1:3) .\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTreatment conditions\u003c/h3\u003e\n\u003cp\u003eTwo PDCA patient-derived pancreatic organoid lines were treated following a previously established protocol: each organoid line was plated in four separate 24-well plates, corresponding to four different treatment conditions:\u003c/p\u003e\u003cp\u003e\u003cspan\u003e1. Control (CTRL): Organoids were not exposed to radiations.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e2. Irradiation on a standard linear accelerator (IR): Organoids were irradiated using a conventional linear accelerator (TrueBeam Edge, Varian) delivering 6FFF MV photon beams at a dose rate of 600 MU/min. A single dose of 10 Gy was delivered using a rectangular anterior\u0026ndash;posterior field, positioning the samples at the isocenter. This dose level was selected to simulate a standard single-fraction stereotactic body radiation therapy (SBRT) session as employed in clinical practice.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe experimental setup is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e3. Irradiation in MRI-LINAC with real-time MRI guidance (IRrtMRI): irradiation was performed with the MRIdian system at 0.35 T and a single dose of 10 Gy, maintaining the same field configuration. Additionally, real-time cine-MRI was acquired during irradiation using a TrueFISP (true fast imaging with steady-state free precession) sequence at 8 frames per second in a sagittal plane. Experimental setup is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eViability assays\u003c/h3\u003e\n\u003cp\u003eDissociated Patient-Derived Organoids (PDOs) were resuspended in 2% BME/growth medium and seeded in 100 \u0026micro;l volume on BME pre-coated 96-well plates after 72 hours. After seven days the viability was determined by CellTiter-Glo\u0026reg; Luminescent Cell Viability Assay (Promega) using a microplate reader (Spark, Tecan). Viability was determined relative to the untreated control cells and analysed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e\n\u003ch3\u003eOrganoid size measurement and classification\u003c/h3\u003e\n\u003cp\u003eThe size of organoids was measured as described in Morrow et al [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A straight line was drawn across the widest part for each organoid to determine its diameter using ImageJ software. This process was repeated until the diameter of each organoid in the sample was measured. To classify organoids by size, the diameter range was first calculated by subtracting the diameter of the smallest organoid from that of the largest, then divided by three. The result determined the range to define small, medium and large organoid categories. Each organoid was subsequently assigned to one of these size groups based on its measured diameter. All measurements were performed in triplicate to ensure reproducibility.\u003c/p\u003e\n\u003ch3\u003eFACS analysis\u003c/h3\u003e\n\u003cp\u003ePDO cells were fixed and stored in 70% ethanol for up to one week. After washing with DPBS, 10\u003csup\u003e6\u003c/sup\u003e cells were stained with 0.3 mL of 10 \u0026micro;g/ml 7-AAD (7-amino-actinomycin D) in DPBS supplemented with protease inhibitors and RNAsin (1:1000). After 30 min of incubation at 4\u0026deg;C in the dark, cells were sorted by DNA content using DxFLEX Beckman Coulter.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eWhole cell proteins were obtained by lysing the organoids with RIPA lysis buffer (60mM Tris-HCl pH 7.5, 1% NP40, 150mM NaCl, 50mM NaF, 2mM EDTA) supplemented with 2mM Na- orthovanadate, 1mM DTT and protease-inhibitors. Lysates were incubated on ice for 30 min, briefly sonicated, and centrifuged (15 min at 15 000 rpm, 4\u0026deg;C). Protein extracts were quantified by Bradford assays, diluted in SDS-sample buffer and boiled at 95\u0026deg; for five min. Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis, blotted to PVDF (Polyvinylidene fluoride), and transferred overnight. After blocking in 5% non-fat milk, membranes were probed with the following primary antibodies: 1:1000 anti- γH2AX (Cell Signalling), 1:1000 anti-PARP (Cell Signalling) and 1:1000 anti\u0026ndash; βTubulin (Cell Signalling), at 4\u0026deg;C, overnight. After incubation with secondary horseradish peroxidase-conjugated antibody, 1:5000 dilution for one hour, proteins were visualized by the enhanced chemiluminescence system (Bio-Rad or Amersham Biosciences, Buckinghamshire, UK) using a ChemiDoc imaging system (UVITEC, Cambridge).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eCell proliferation, apoptosis and western blot data were analysed by one-way analysis of variance (ANOVA), using Tukey\u0026rsquo;s multiple comparison test to determine if significant differences existed between groups. PDO size data were evaluated by the two-way analysis of variance (ANOVA), using Bonferroni\u0026rsquo;s multiple comparisons test. All data were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;\u0026ge;\u0026thinsp;3) and statistical analyses were performed using the GraphPad Prism10 Software. A p-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthics statement\u003c/h3\u003e\n\u003cp\u003eHuman pancreatic ductal adenocarcinoma (PDAC) tissues used for the generation of patient-derived organoids were obtained from treatment-na\u0026iuml;ve patients undergoing biopsy at Fondazione Policlinico Universitario \u0026ldquo;A. Gemelli\u0026rdquo; IRCCS (Rome, Italy). All experimental procedures involving human biological samples were performed in accordance with relevant guidelines and regulations and with the Declaration of Helsinki. The study protocol was approved by the Institutional Ethics Committee of Fondazione Policlinico Universitario \u0026ldquo;A. Gemelli\u0026rdquo; IRCCS. Written informed consent was obtained from all patients prior to sample collection.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eTwo PDAC PDOs, PDAC-9 and PDAC-12, were established from treatment-na\u0026iuml;ve patients and preserved genetic phenotypes of the corresponding tissues, as previously reported [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo evaluate the impact of irradiation in combination with real-time cine-MRI on PDAC organoid growth \u003cem\u003eex vivo\u003c/em\u003e, the study comprised three experimental groups (see M\u0026amp;M for details):\u003c/p\u003e \u003cp\u003e\u003cspan\u003e1. Control (CTRL): PDO were not exposed to radiations.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e2. Irradiation of PDO on a standard linear accelerator (IR): experimental setup shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e3. Irradiation in MRI-LINAC with real-time MRI guidance (IRrtMRI): experimental setup is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eTo directly assess the impact of treatments on PDAC organoid growth \u003cem\u003eex vivo\u003c/em\u003e, PDOs were γ-irradiated with 10 Gy and analysed after 72 h from irradiation as illustrated in the scheme of the study workflow (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Morphological evaluation of the PDAC-9 and PDAC-12 PDOs, grown in 3D cultures for 13 days after treatments, indicated that both irradiations alone and its IRrtMRI combination caused a reduction of proliferation as shown in representative brightfield images in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003eTo directly assess the impact of treatments on the self-renewal ability of PDOs, we passaged them after 72 h from irradiation and measured the size of the newly formed organoids after additional seven days. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, PDAC-9 and PDAC-12 exhibited a significant reduction in the number and size of the organoids formed after treatment, showing a higher impact of the IRrtMRI combination exposure. A quantitative evaluation of this effect was carried out by measuring the size of the organoids, classified in three groups small, medium and large. For both PDAC-9 and PDAC-12 organoids, the percentage of the \u0026ldquo;large size\u0026rdquo; subgroup dropped to zero after both IR and IRrtMRI treatments, while the percentage of \u0026ldquo;small size\u0026rdquo; PDOs increased concomitantly with greater effect in IRrtMRI combination treatment. For the \u0026ldquo;medium size\u0026rdquo; subgroup, the greater inhibitory effect was shown after IRrtMRI treatment for both PDOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eImportantly, the result of the morphological analysis was in line with the data of the viability assay, which showed that the combined IRrtMRI treatment significantly enhanced the inhibitory effect of IR on organoid cell viability in both PDO lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo determine if the inhibition of cell viability is due to reduced growth of PDOs accompanied by cell death, we also performed 7-AAD staining to assess the DNA content in PDOs at 72h after irradiation.\u003c/p\u003e \u003cp\u003eFlow cytometry analysis after 7-AAD staining showed that 40% of the cells of the PDAC-12 PDO were apoptotic (sub-G1 phase DNA content) and this effect was significantly increased to 70% in PDOs exposed to the IRrtMRI combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In contrast, no significant effect of these treatments on cell death was observed in PDAC-9.\u003c/p\u003e \u003cp\u003eThese results were confirmed by western blot analysis of cleaved-PARP1 protein, a marker of apoptosis, which was only induced in PDAC-12 cells after IRrtMRI combined treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On the other hand, the phosphorylation of γH2AX, a marker of DNA damage, was significantly increased in both PDO lines exposed to the combined IRrtMRI treatment compared to IR alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn conclusion, these data indicate that concurrent exposure to IR and rtMRI exerts a stronger antiproliferative effect on PDAC PDOs in comparison to IR treatment alone, suggesting that the combination treatment enhances the inhibitory effects of IR in PDAC PDOs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results obtained on two pancreatic ductal adenocarcinoma PDOs, PDAC-9 and PDCA-12, indicate that irradiation in the presence of a SMF, combined with real-time MRI acquisition, induces a stronger inhibition of cell growth than irradiation alone, intensifying genotoxic damage and promoting apoptosis, as demonstrated by the increased phosphorylation of γH2AX and the induction of cleaved PARP.\u003c/p\u003e \u003cp\u003eFour mechanisms described in the literature provide a useful interpretative framework for these findings.\u003c/p\u003e \u003cp\u003eFirstly, static magnetic fields can prolong the lifetime of free radicals in the triplet state, thereby extending the time window for interactions with DNA and amplifying radiation-induced damage. This mechanism, reported by the UCLA group [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], has been associated with increased cellular lethality both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e under weak magnetic fields. In this context, the enhanced γH2AX phosphorylation and apoptotic response observed in our experiments are consistent with the hypothesis that the static magnetic field acts as a physical co-factor capable of potentiating the effects of radiotherapy.\u003c/p\u003e \u003cp\u003eSecondly, the opposite effect has been reported in X-ray\u0026ndash;guided radiotherapy systems, where low-dose imaging can trigger an adaptive response mediated by survivin, leading to tumor radioprotection. This phenomenon, described by the Chicago group [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], suggests that imaging modality can influence tumor biology independently of the therapeutic irradiation. In our model, the use of MRIgRT eliminates X-ray exposure during imaging and thus reduces the likelihood of inducing such a radioprotective response, providing an additional biological advantage that complements the potential radiosensitizing effect of the magnetic field.\u003c/p\u003e \u003cp\u003eThirdly, static magnetic fields have been reported to induce partial orientation or polarization of DNA due to the diamagnetic anisotropy of nucleic acid bases. This phenomenon, first described by Maret et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and later elaborated by Vavrinsk\u0026aacute; et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], can increase the surface area of DNA exposed to radiation damage while simultaneously interfering with the accessibility of DNA repair proteins, thereby delaying the resolution of double-strand breaks. In such context, in PDCA-9 and PDCA-12 the observed increased phosphorylation of γH2AX could be the result of both initial DNA damage and impaired or delayed DNA repair.\u003c/p\u003e \u003cp\u003eFourth, Wishart et al. demonstrated that the presence of a moderate-intensity static magnetic field (1.5 T) significantly enhanced X-ray-induced apoptosis in a three-dimensional pancreatic cancer model, with effects particularly pronounced under hypoxic conditions that closely resemble the pancreatic tumor microenvironment [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These results suggest that the magnetic field intrinsic to MRIgRT systems may contribute to biological radiosensitization beyond physical dose delivery. In addition, earlier in vitro studies summarized by Dini and Abbro reported that static magnetic fields of moderate intensity can modulate key cellular processes, including proliferation, apoptosis, ion fluxes, and oxidative stress responses, in a cell-type- and context-dependent manner [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these possible explanations, it is important to note that the existing literature reports conflicting results. Studies conducted at 1 Tesla [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] or using the Unity platform did not find significant differences in biological response to the combination of radiotherapy and SMF, whereas other experimental observations reported radiosensitizing effects attributable to the magnetic field [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Some preliminary reports have even suggested that sample heating during MRI acquisition may act as a confounding factor. Our investigation, based on the use of PDOs, represents an innovative approach compared with conventional experimental radiobiological models. PDOs are three-dimensional structures generated directly from patient tumor samples that faithfully recapitulate the histological architecture of the original tissue. This includes the spatial organization of cells, cell\u0026ndash;cell and cell\u0026ndash;matrix interactions, as well as gradients of nutrients, oxygen, and growth factors. Unlike traditional two-dimensional (2D) culture systems, in which homogeneous cell lines are grown on flat artificial surfaces, PDOs preserve the phenotypic heterogeneity of the primary tumor, providing a more accurate reflection of intratumoral diversity and therapeutic response. This model is particularly effective for the generation of experimental data that are physiologically more relevant and predictive, thereby improving the translational potential of preclinical studies and their applicability to clinical settings [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, our study provides an original contribution by exploiting the unique features of PDOs to investigate biological mechanisms and treatment responses in a pre-clinical model that more closely mirrors patient tumor biology than conventional 2D cultures.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates that the combination of irradiation, static magnetic fields, and real-time MRI acquisition intensifies DNA damage and apoptotic activation in pancreatic cancer\u0026ndash;derived organoids. Our findings can be interpreted in light of three mechanisms: the prolonged lifetime of free radicals in the presence of magnetic fields, which amplifies genotoxic damage [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; the absence of X-ray positioning imaging acquired seconds to minutes before treatment fraction delivery, which eliminates the possibility of a survivin-mediated adaptive radioprotective response [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; and magnetic field\u0026ndash;induced DNA polarization, which may increase DNA exposure to damage and interfere with repair processes [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis tripartite interpretative framework suggests that MRIgRT may not only offer technical advantages in unprecedented target volume visualization and online adaptation, but it could also confer an intrinsic biological benefit compared with X-ray\u0026ndash;guided radiotherapy. Our work provides a robust proof of principle supporting the value of PDOs as a platform for investigating innovative combinations of magnetic fields and radiotherapy.\u003c/p\u003e \u003cp\u003eHowever, the different responses observed among two PDO lines, probably due to the intrinsic genetic and molecular features or interpatient heterogeneity, underscores the need for further studies in a larger cohort of PDOs to identify the most responsive patient subgroups and the specific contributions of radical-mediated effects, DNA polarization, imaging-related mechanisms, and potential confounding factors such as MRI-induced heating. Moreover, further research is required to understand the specific mechanisms underlying the stronger antiproliferative effect of IRrtMRI combination exposure and demonstrate its selective effect on pancreatic tumor cells in comparison to normal cells.\u003c/p\u003e \u003cp\u003eOverall, the data presented here strengthens the biological rationale for the development and clinical validation of MR-linac based therapeutic protocols, potentially improving radiotherapeutic outcomes for pancreatic cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the support of Prof. Michael Chuong from Miami Cancer Institute, Miami, Florida (USA) in reviewing this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResearch funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare support of Radius srl of Budrio (BO), Italy, for funding these experiments.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.B. and C.G.C. conceived and designed the study and supervised the overall research project.\u003c/p\u003e\n\u003cp\u003eL.B., C.G.C., L.P., G.C., M.N., M.G., A.R., and G.P. contributed to radiotherapy planning, experimental irradiation procedures, data acquisition, and interpretation of radiobiological results.\u003c/p\u003e\n\u003cp\u003eA.C., C.C., E.C., and R.V. established and maintained the patient-derived organoid models and performed organoid-based experiments and analyses.\u003c/p\u003e\n\u003cp\u003eR.V. and C.S. contributed to the biological interpretation of the data and to the analysis of molecular and cellular endpoints.\u003c/p\u003e\n\u003cp\u003eK.B. provided critical expertise in radiobiology and contributed to data interpretation and manuscript revision.\u003c/p\u003e\n\u003cp\u003eR.M., V.T., S.A., and G.T. contributed to patient recruitment, clinical characterization, and provided clinical and translational input relevant to pancreatic cancer.\u003c/p\u003e\n\u003cp\u003eL.B., C.G.C., and M.A.G. drafted the manuscript. All authors contributed to critical revision of the manuscript for important intellectual content.\u003c/p\u003e\n\u003cp\u003eAll authors approved the final version of the manuscript and agree to be accountable for their own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCapurso, G. \u0026amp; Sette, C. Drug resistance in pancreatic cancer: New player caught in act. \u003cem\u003eEBioMedicine\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e, 39\u0026ndash;40. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ebiom.2019.02.008\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2019.02.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHidalgo, M. Pancreatic cancer. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e362\u003c/b\u003e, 1605\u0026ndash;1617. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMra0901557\u003c/span\u003e\u003cspan address=\"10.1056/NEJMra0901557\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed, S., Van Buren, G. \u0026amp; Fisher, W. E. Pancreatic cancer: advances in treatment. \u003cem\u003eWorld J. Gastroenterol.\u003c/em\u003e \u003cb\u003e20\u003c/b\u003e, 9354\u0026ndash;9360. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3748/wjg.v20.i28.9354\u003c/span\u003e\u003cspan address=\"10.3748/wjg.v20.i28.9354\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLominska, C. E., Unger, K., Nasr, N. M., Haddad, N. \u0026amp; Gagnon, G. Stereotactic body radiation therapy for reirradiation of localized adenocarcinoma of the pancreas. \u003cem\u003eRadiat. Oncol.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 74. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1748-717X-7-74\u003c/span\u003e\u003cspan address=\"10.1186/1748-717X-7-74\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFranzone, P. et al. Image-guided radiation therapy (IGRT): practical recommendations of Italian Association of Radiation Oncology (AIRO). \u003cem\u003eRadiol. Med.\u003c/em\u003e \u003cb\u003e121\u003c/b\u003e, 958\u0026ndash;965. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11547-016-0674-x\u003c/span\u003e\u003cspan address=\"10.1007/s11547-016-0674-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChin, S. et al. Magnetic resonance-guided radiation therapy: A review. \u003cem\u003eJ. Med. Imaging Radiat. Oncol.\u003c/em\u003e \u003cb\u003e64\u003c/b\u003e, 163\u0026ndash;177. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1754-9485.12968\u003c/span\u003e\u003cspan address=\"10.1111/1754-9485.12968\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoldrini, L. et al. Online adaptive magnetic resonance guided radiotherapy for pancreatic cancer: state of the art, pearls and pitfalls. \u003cem\u003eRadiat. Oncol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 71. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13014-019-1275-3\u003c/span\u003e\u003cspan address=\"10.1186/s13014-019-1275-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlacidi, L. et al. On-line adaptive MR guided radiotherapy for locally advanced pancreatic cancer: Clinical and dosimetric considerations. \u003cem\u003eTech. Innov. Patient Support Radiat. Oncol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 15\u0026ndash;21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tipsro.2020.06.001\u003c/span\u003e\u003cspan address=\"10.1016/j.tipsro.2020.06.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorradini, S. et al. MR-guidance in clinical reality: current treatment challenges and future perspectives. \u003cem\u003eRadiat. Oncol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 92. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13014-019-1308-y\u003c/span\u003e\u003cspan address=\"10.1186/s13014-019-1308-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKl\u0026uuml;ter, S. Technical design and concept of a 0.35 T MR-Linac. \u003cem\u003eClin. Transl Radiat. Oncol.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e, 98\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ctro.2019.04.007\u003c/span\u003e\u003cspan address=\"10.1016/j.ctro.2019.04.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaaymakers, B. W. et al. Integrating a 1.5 T MRI scanner with a 6 MV accelerator: proof of concept. \u003cem\u003ePhys. Med. Biol.\u003c/em\u003e \u003cb\u003e54\u003c/b\u003e, N229\u0026ndash;237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1088/0031-9155/54/12/N01\u003c/span\u003e\u003cspan address=\"10.1088/0031-9155/54/12/N01\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohajer, J. K., Nisbet, A., Velliou, E., Ajaz, M. \u0026amp; Schettino, G. Biological effects of static magnetic field exposure in the context of MR-guided radiotherapy. \u003cem\u003eBr. J. Radiol.\u003c/em\u003e \u003cb\u003e92\u003c/b\u003e, 20180484. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1259/bjr.20180484\u003c/span\u003e\u003cspan address=\"10.1259/bjr.20180484\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWishart, G., Gupta, P., Nisbet, A., Velliou, E. \u0026amp; Schettino, G. Enhanced effect of X-rays in the presence of a static magnetic field within a 3D pancreatic cancer model. \u003cem\u003eBr. J. Radiol.\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e (1143), 20220832. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1259/bjr.20220832\u003c/span\u003e\u003cspan address=\"10.1259/bjr.20220832\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023). Epub 2023 Jan 14. PMID: 36475863; PMCID: PMC9975369.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDini, L. \u0026amp; Abbro, L. Bioeffects of moderate-intensity static magnetic fields on cell cultures. \u003cem\u003eMicron\u003c/em\u003e \u003cb\u003e36\u003c/b\u003e (3), 195\u0026ndash;217. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.micron.2004.12.009\u003c/span\u003e\u003cspan address=\"10.1016/j.micron.2004.12.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2005). Epub 2005 Jan 25. PMID: 15725590.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFang, Z., Li, P., Du, F., Shang, L. \u0026amp; Li, L. The role of organoids in cancer research. \u003cem\u003eExp. Hematol. Oncol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40164-023-00433-y\u003c/span\u003e\u003cspan address=\"10.1186/s40164-023-00433-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEke, I. \u0026amp; Cordes, N. Radiobiology goes 3D: how ECM and cell morphology impact on cell survival after irradiation. \u003cem\u003eRadiother Oncol.\u003c/em\u003e \u003cb\u003e99\u003c/b\u003e, 271\u0026ndash;278. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.radonc.2011.06.007\u003c/span\u003e\u003cspan address=\"10.1016/j.radonc.2011.06.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, J., Wang, K., Tang, Y. \u0026amp; Zheng, D. Applications and perspectives of tumor organoids in radiobiology (Review). \u003cem\u003eOncol. Rep.\u003c/em\u003e \u003cb\u003e52\u003c/b\u003e, 100. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/or.2024.8759\u003c/span\u003e\u003cspan address=\"10.3892/or.2024.8759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDriehuis, E. et al. Pancreatic cancer organoids recapitulate disease and allow personalized drug screening. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e116\u003c/b\u003e, 26580\u0026ndash;26590. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1911273116\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1911273116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicosia, L. et al. Combinatorial Effect of Magnetic Field and Radiotherapy in PDAC Organoids: A Pilot Study. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 609. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biomedicines8120609\u003c/span\u003e\u003cspan address=\"10.3390/biomedicines8120609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuta, V. et al. An alternative splicing signature defines the basal-like phenotype and predicts worse clinical outcome in pancreatic cancer. \u003cem\u003eCell. Rep. Med.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e (2), 101411. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.xcrm.2024.101411\u003c/span\u003e\u003cspan address=\"10.1016/j.xcrm.2024.101411\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024). Epub 2024 Feb 6. PMID: 38325381; PMCID: PMC10897606.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrow, R. J., Ernst, M. \u0026amp; Poh, A. R. Longitudinal quantification of mouse gastric tumor organoid viability and growth using luminescence and microscopy. \u003cem\u003eSTAR. Protoc.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 102110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.xpro.2023.102110\u003c/span\u003e\u003cspan address=\"10.1016/j.xpro.2023.102110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwamoto, K. S. et al. Weak Magnetic Fields Enhance the Efficacy of Radiation Therapy. \u003cem\u003eAdv. Radiat. Oncol.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 100645. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.adro.2021.100645\u003c/span\u003e\u003cspan address=\"10.1016/j.adro.2021.100645\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrdina, D. J. et al. A survivin-associated adaptive response in radiation therapy. \u003cem\u003eCancer Res.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 4418\u0026ndash;4428. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1158/0008-5472.CAN-12-4640\u003c/span\u003e\u003cspan address=\"10.1158/0008-5472.CAN-12-4640\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaret, G. Orientation of Nucleic Acids in High Magnetic Fields. \u003cem\u003ePhys. Rev. Lett.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e, 397\u0026ndash;400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1103/PhysRevLett.35.397\u003c/span\u003e\u003cspan address=\"10.1103/PhysRevLett.35.397\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1975).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVavrinsk\u0026aacute;, A. et al. Impact of nucleic acid self-alignment in a strong magnetic field on the interpretation of indirect spin-spin interactions. \u003cem\u003eJ. Biomol. NMR\u003c/em\u003e. \u003cb\u003e64\u003c/b\u003e, 53\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10858-015-0005-x\u003c/span\u003e\u003cspan address=\"10.1007/s10858-015-0005-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9009316/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9009316/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurpose/Objective\u003c/p\u003e \u003cp\u003ePancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies, with limited treatment options. Magnetic Resonance-guided Radiotherapy (MRIgRT) combines real-time imaging and irradiation under a static magnetic field (SMF), potentially influencing biological responses beyond its technical advantages. This study aimed to investigate the combined effects of SMF and radiotherapy on patient-derived PDAC organoids (PDOs) to assess whether MRI guidance modulates radiation-induced cytotoxicity.\u003c/p\u003e \u003cp\u003eMaterial/Methods\u003c/p\u003e \u003cp\u003eTwo PDO lines (PDAC-9 and PDAC-12) were established from treatment-na\u0026iuml;ve PDAC tissues and cultured in 3D BME matrices. Organoids were exposed to three experimental conditions: (i) untreated control (CTRL), (ii) 10 Gy irradiation using a standard linac (IR), and (iii) 10 Gy irradiation on a 0.35 T MR-Linac with real-time cine-MRI acquisition (IRrtMRI). Organoid viability was assessed by CellTiter-Glo luminescence assay. Morphometric analysis was performed using Bright-field images classifying organoids as small, medium, or large. Cell death was quantified by 7-AAD flow cytometry, while DNA damage and apoptosis were evaluated via western blotting for γH2AX and cleaved-PARP1. Statistical analysis was performed by ANOVA with multiple comparisons (GraphPad Prism 10).\u003c/p\u003e \u003cp\u003eResults\u003c/p\u003e \u003cp\u003eBoth PDO lines exhibited reduced growth and viability after irradiation, with a significantly stronger effect under combined IRrtMRI exposure. The proportion of large organoids decreased to 0%, while small organoids markedly increased in both PDAC-9 and PDAC-12 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Cell viability assays confirmed enhanced inhibition following IRrtMRI compared to IR alone (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Flow cytometry revealed that apoptotic cells increased from 40% (IR) to 70% (IRrtMRI) in PDAC-12, whereas PDAC-9 showed relative resistance. Western blot analysis demonstrated increased γH2AX phosphorylation in both PDOs and cleaved-PARP1 induction selectively in PDAC-12 under combined treatment, indicating augmented DNA damage and apoptosis.\u003c/p\u003e \u003cp\u003eConclusion\u003c/p\u003e \u003cp\u003eExposure to radiotherapy under a static 0.35 T magnetic field with real-time MRI enhances genotoxic and apoptotic effects in PDAC organoids compared to conventional irradiation. The observed radiosensitization may derive from magnetic field\u0026ndash;mediated prolongation of radical lifetimes, absence of CBCT X-ray\u0026ndash;induced adaptive responses, and DNA polarization phenomena. These findings suggest a possible biological advantage of MRIgRT beyond improved targeting accuracy, supporting further investigation into its translational and clinical implications in pancreatic cancer.\u003c/p\u003e","manuscriptTitle":"Increased cytotoxic effects via combined treatment of magnetic field and radiation therapy in patient-derived organoids of pancreatic ductal adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-19 16:25:11","doi":"10.21203/rs.3.rs-9009316/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e9e33d1-1825-47fb-b0aa-e1a3749b27fa","owner":[],"postedDate":"March 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":64650995,"name":"Biological sciences/Cancer"},{"id":64650996,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-04-08T05:56:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-19 16:25:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9009316","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9009316","identity":"rs-9009316","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}