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Radiotherapy represents a major curative treatment for prostate cancer (PCa), but some patients will develop radioresistance (RR) and relapse. The underlying mechanisms remain poorly understood, and miRNAs might be key players in the acquisition and maintenance of RR. Through their encapsulation in small extracellular vesicles (EVs), they can also be relevant biomarkers of radiation response. Using next-generation sequencing, we found that miR-200c-3p was downregulated in PCa RR cells and in their small EVs due to a gain of methylation on its promoter during RR acquisition. We next showed that its exogenous overexpression restores the radiosensitivity of RR cells by delaying DNA repair through the targeting of HP1α. Interestingly, we also observed downregulation of miR-200c-3p expression by DNA methylation in radiation-resistant lung and breast cancer cell lines. In summary, our study demonstrates that the downregulation of miR-200c-3p expression in PCa cells and in their small EVs could help distinguish radioresistant from sensitive tumor cells. This miRNA targets HP1α to delay DNA repair and promote cell death., (© 2024. The Author(s).)

As part of translational research projects, mice may be irradiated on radiobiology platforms such as the one at the ARRONAX cyclotron. Generally, these platforms do not feature an integrated imaging system. Moreover, in the context of ultra-high dose-rate radiotherapy (FLASH-RT), treatment planning should consider potential changes in the beam characteristics and internal movements in the animal. A patient-like set-up and methodology has been implemented to ensure target coverage during conformal irradiations of the brain, lungs and intestines. In addition, respiratory cycle amplitudes were quantified by fluoroscopic acquisitions on a mouse, to ensure organ coverage and to assess the impact of respiration during FLASH-RT using the 4D digital phantom MOBY. Furthermore, beam incidence direction was studied from mice µCBCT and Monte Carlo simulations. Finally,in vivodosimetry with dose-rate independent radiochromic films (OC-1) and their LET dependency were investigated. The immobilization system ensures that the animal is held in a safe and suitable position. The geometrical evaluation of organ coverage, after the addition of the margins around the organs, was satisfactory. Moreover, no measured differences were found between CONV and FLASH beams enabling a single model of the beamline for all planning studies. Finally, the LET-dependency of the OC-1 film was determined and experimentally verified with phantoms, as well as the feasibility of using these filmsin vivoto validate the targeting. The methodology developed ensures accurate and reproducible preclinical irradiations in CONV and FLASH-RT without in-room image guidance in terms of positioning, dose calculation andin vivodosimetry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Associazione Italiana di Fisica Medica e Sanitaria. Published by Elsevier Ltd. All rights reserved.)

The ability to reduce toxicity of ultra-high dose rate (UHDR) helium ion irradiation has not been reported in vivo. Here, we tested UHDR helium ion irradiation in an embryonic zebrafish model. Our results show that UHDR helium ions spare body development and reduce spine curvature, compared to conventional dose rate., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: Recently, ultrahigh-dose-rate radiation therapy (UHDR-RT) has emerged as a promising strategy to increase the benefit/risk ratio of external RT. Extensive work is on the way to characterize the physical and biological parameters that control the so-called "Flash" effect. However, this healthy/tumor differential effect is observable in in vivo models, which thereby drastically limits the amount of work that is achievable in a timely manner., Methods and Materials: In this study, zebrafish embryos were used to compare the effect of UHDR irradiation (8-9 kGy/s) to conventional RT dose rate (0.2 Gy/s) with a 68 MeV proton beam. Viability, body length, spine curvature, and pericardial edema were measured 4 days postirradiation., Results: We show that body length is significantly greater after UHDR-RT compared with conventional RT by 180 µm at 30 Gy and 90 µm at 40 Gy, while pericardial edema is only reduced at 30 Gy. No differences were obtained in terms of survival or spine curvature., Conclusions: Zebrafish embryo length appears as a robust endpoint, and we anticipate that this model will substantially fasten the study of UHDR proton-beam parameters necessary for "Flash.", (© 2022 The Authors.)

During ultra-high dose rate (UHDR) external radiation therapy, healthy tissues appear to be spared while tumor control remains the same compared to conventional dose rate. However, the understanding of radiochemical and biological mechanisms involved are still to be discussed. This study shows how the hydrogen peroxide (H2O2) production, one of the reactive oxygen species (ROS), could be controlled by early heterogenous radiolysis processes in water during UHDR proton-beam irradiations. Pure water was irradiated in the plateau region (track-segment) with 68 MeV protons under conventional (0.2 Gy/s) and several UHDR conditions (40 Gy/s to 60 kGy/s) at the ARRONAX cyclotron. Production of H2O2 was then monitored using the Ghormley triiodide method. New values of GTS(H2O2) were added in conventional dose rate. A substantial decrease in H2O2 production was observed from 0.2 to 1.5 kGy/s with a more dramatic decrease below 100 Gy/ s. At higher dose rate, up to 60 kGy/s, the H2O2 production stayed stable with a mean decrease of 38% ± 4%. This finding, associated to the decrease in the production of hydroxyl radical (•OH) already observed in other studies in similar conditions can be explained by the well-known spur theory in radiation chemistry. Thus, a two-step FLASH-RT mechanism can be envisioned: an early step at the microsecond scale mainly controlled by heterogenous radiolysis, and a second, slower, dominated by O2 depletion and biochemical processes. To validate this hypothesis, more measurements of radiolytic species will soon be performed, including radicals and associated lifetimes., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

Predicting a response of osteosarcoma patients to chemotherapy, such as doxorubicin or high-dose methotrexate cocktail, remains a challenge in the clinic. Moreover, the prognostic value of currently used necrosis analysis is debatable. New markers of the therapeutic response or the prognostic response are urgently needed. The microenvironment plays a key role in the vascularization of highly heterogeneous tumors. Using the syngeneic MOS-J mouse model of osteosarcoma, we focused our study on the immunohistochemistry of tumor vascularization in order to identify new vessel markers, and to search for potential markers of the therapeutic response. Endomucin+, CD31+, and α-SMA+-positive elements were quantified in control (n=6) and doxorubicin-treated (n=6) mice in three different intra-tumor locations. We also used co-labeling to assess CD31+/Endomucin+ and CD31+/α-SMA+ co-expression. We identified a central tumor zone with a low vascularization profile for all of these markers. We identified two distinct types of vessels: CD31+/Endomucin+ vessels with a sprouting, neo-angiogenic, interlaced appearance, and CD31+/α-SMA+ vessel with a well-defined, mature structure. Doxorubicin appeared to reduce CD31+ expression in the tumor invasion front. In the doxorubicin-sensitive model, there were four times more CD31+/α-SMA+ elements than in the poorly responsive model. Therefore, we propose a methodology based on immunohistochemistry and multiplexed immunofluorescence to use endomucin as a promising new vascular marker in the osteosarcoma model. Moreover, our results suggest that CD31+/α-SMA+ vessels could be considered to be indicators of vasculature normalization and they may be used as specific markers of a good therapeutic response., Competing Interests: None., (AJCR Copyright © 2022.)

Purpose: The ARRONAX cyclotron facility offers the possibility to deliver proton beams from low to ultra-high dose rates (UHDR). As a good control of the dosimetry is a prerequisite of UHDR experimentations, we evaluated in different conditions the usability and the dose rate dependency of several radiochromic films commonly used for dosimetry in radiotherapy., Methods: We compared the dose rate dependency of three types of radiochromic films: GAFchromic™ EBT3 and GAFchromic™ EBT-XD (Ashland Inc., Wayne, NJ, USA), and OrthoChromic OC-1 (OrthoChrome Inc., Hillsborough, NJ, USA), after proton irradiations at various mean dose rates (0.25, 40, 1500, and 7500 Gy/s) and for 10 doses (2-130 Gy). We also evaluated the dose rate dependency of each film considering beam structures, from single pulse to multiple pulses with various frequencies., Results: EBT3 and EBT-XD films showed differences of response between conventional (0.25 Gy/s) and UHDR (7500 Gy/s) conditions, above 10 Gy. On the contrary, OC-1 films did not present overall difference of response for doses except below 3 Gy. We observed an increase of the netOD with the mean dose rate for EBT3 and EBT-XD films. OC-1 films did not show any impact of the mean dose rate up to 7500 Gy/s, above 3 Gy. No difference was found based on the beam structure, for all three types of films., Conclusions: EBT3 and EBT-XD radiochromic films should be used with caution for the dosimetry of UHDR proton beams over 10 Gy. Their overresponse, which increases with mean dose rate and dose, could lead to non-negligible overestimations of the absolute dose. OC-1 films are dose rate independent up to 7500 Gy/s in proton beams. Films response is not impacted by the beam structure. A broader investigation of the usability of OC-1 films in UHDR conditions should be conducted at intermediate and higher mean dose rates and other beam energies., (© 2022 American Association of Physicists in Medicine.)

Physical activity is increasingly recognized as a strategy able to improve cancer patient outcome, and its potential to enhance treatment response is promising, despite being unclear. In our study we used a preclinical model of prostate cancer to investigate whether voluntary wheel running (VWR) could improve tumor perfusion and enhance radiotherapy (RT) efficiency. Nude athymic mice were injected with PC-3 cancer cells and either remained inactive or were housed with running wheels. Apparent microbubble transport was enhanced with VWR, which we hypothesized could improve the RT response. When repeating the experiments and adding RT, however, we observed that VWR did not influence RT efficiency. These findings contrasted with previous results and prompted us to evaluate if the lack of effects observed on tumor growth could be attributable to the physical activity modality used. Using PC-3 and PPC-1 xenografts, we randomized mice to either inactive controls, VWR, or treadmill running (TR). In both models, TR (but not VWR) slowed down tumor growth, suggesting that the anti-cancer effects of physical activity are dependent on its modalities. Providing a better understanding of which activity type should be recommended to cancer patients thus appears essential to improve treatment outcomes.

Prostate cancer is the most frequently diagnosed cancer in men and a leading cause of cancer-related death. In recent decades, the development of immunotherapies has resulted in great promise to cure metastatic disease. However, prostate cancer has failed to show any significant response, presumably due to its immunosuppressive microenvironment. There is therefore growing interest in combining immunotherapy with other therapies able to relieve the immunosuppressive microenvironment. Radiation therapy remains the mainstay treatment for prostate cancer patients, is known to exhibit immunomodulatory effects, depending on the dose, and is a potent inducer of immunogenic tumor cell death. Optimal doses of radiotherapy are thus expected to unleash the full potential of immunotherapy, improving primary target destruction with further hope of inducing immune-cell-mediated elimination of metastases at distance from the irradiated site. In this review, we summarize the current knowledge on both the tumor immune microenvironment in prostate cancer and the effects of radiotherapy on it, as well as on the use of immunotherapy. In addition, we discuss the utility to combine immunotherapy and radiotherapy to treat oligometastatic metastatic prostate cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ollivier, Labbé, Fradin, Potiron and Supiot.)

Proton therapy (PRT) is an irradiation technique that aims at limiting normal tissue damage while maintaining the tumor response. To study its specificities, the ARRONAX cyclotron is currently developing a preclinical structure compatible with biological experiments. A prerequisite is to identify and control uncertainties on the ARRONAX beamline, which can lead to significant biases in the observed biological results and dose-response relationships, as for any facility. This paper summarizes and quantifies the impact of uncertainty on proton range, absorbed dose, and dose homogeneity in a preclinical context of cell or small animal irradiation on the Bragg curve, using Monte Carlo simulations. All possible sources of uncertainty were investigated and discussed independently. Those with a significant impact were identified, and protocols were established to reduce their consequences. Overall, the uncertainties evaluated were similar to those from clinical practice and are considered compatible with the performance of radiobiological experiments, as well as the study of dose-response relationships on this proton beam. Another conclusion of this study is that Monte Carlo simulations can be used to help build preclinical lines in other setups.

As the most frequently diagnosed non-skin cancer in men and a leading cause of cancer-related death, understanding the molecular mechanisms that drive treatment resistance in prostate cancer poses a significant clinical need. Radiotherapy is one of the most widely used treatments for prostate cancer, along with surgery, hormone therapy, and chemotherapy. However, inherent radioresistance of tumor cells can reduce local control and ultimately lead to poor patient outcomes, such as recurrence, metastasis and death. The underlying mechanisms of radioresistance have not been fully elucidated, but it has been suggested that miRNAs play a critical role. miRNAs are small non-coding RNAs that regulate gene expression in every signaling pathway of the cell, with one miRNA often having multiple targets. By fine-tuning gene expression, miRNAs are important players in modulating DNA damage response, cell death, tumor aggression and the tumor microenvironment, and can ultimately affect a tumor's response to radiotherapy. Furthermore, much interest has focused on miRNAs found in biofluids and their potential utility in various clinical applications. In this review, we summarize the current knowledge on miRNA deregulation after irradiation and the associated functional outcomes, with a focus on prostate cancer. In addition, we discuss the utility of circulating miRNAs as non-invasive biomarkers to diagnose, predict response to treatment, and prognosticate patient outcomes.

Aim: We here hypothesized that tumor-derived exosomal miRNA (TexomiR) released from irradiated tumors may play a role in the tumor cells escape to natural killer (NK) cells. Materials & methods: Our study included the use of different cancer cell lines, blood biopsies of xenograph mice model and patients treated with radiotherapy. Results: The irradiation of cancer cells promotes the TET2-mediated demethylation of miR-378 promoter, miR-378a-3p overexpression and its loading in exosomes, inducing the decrease of granzyme-B (GZMB) secretion by NK cells. An inverse correlation between TexomiR-378a-3p and GZMB was observed in murine and human blood samples. Conclusion: Our work identifies TexomiR-378a-3p as a molecular signature associated with the loss of NK cells cytotoxicity via the decrease of GZMB expression upon radiotherapy.

Background. The tumor vasculature acts as an interface for the primary tumor. It regulates oxygenation, nutrient delivery, and treatment efficacy including radiotherapy. The response of the tumor vasculature to different radiation doses has been disparately reported. Whereas high single doses can induce endothelial cell death, improved vascular functionality has also been described in a various dose range, and few attempts have been made to reconcile these findings. Therefore, we aimed at comparing the effects of different radiation fractionation regimens on the tumor vascular microenvironment., Methods: Lewis lung and prostate PC3 carcinoma-derived tumors were irradiated with regimens of 10 × 2 Gy, 6 × 4 Gy, 3 × 8 Gy or 2 × 12 Gy fractions. The tumor vasculature phenotype and function was evaluated by immunohistochemistry for endothelial cells (CD31), pericytes (desmin, α-SMA), hypoxia (pimonidazole) and perfusion (Hoechst 33342)., Results: Radiotherapy increased vascular coverage similarly in all fractionation regimens in both models. Vessel density appeared unaffected. In PC3 tumors, hypoxia was decreased and perfusion was enhanced in proportion with the dose per fraction. In LLC tumors, no functional changes were observed at t = 15 days, but increased perfusion was noticed earlier ( t = 9-11 days)., Conclusion: The vascular microenvironment response of prostate and lung cancers to radiotherapy consists of both tumor/dose-independent vascular maturation and tumor-dependent functional parameters., Competing Interests: The authors declare no conflict of interest.

The tumor microenvironment regulates cancer initiation, progression and response to treatment. In particular, the immature tumor vasculature may impede drugs from reaching tumor cells at a lethal concentration. We and others have shown that radiation therapy (RT) induces pericyte recruitment, resembling vascular normalization. Here, we asked whether radiation-induced vascular remodeling translates into improved tissue distribution and efficacy of chemotherapy. First, RT induced vascular remodeling, accompanied by decreased hypoxia and/or increased Hoechst perfusion in prostate PC3 and LNCaP and Lewis lung carcinoma. These results were independent of the RT regimen, respectively 10 × 2 Gy and 2 × 12 Gy, suggesting a common effect. Next, using doxorubicin as a fluorescent reporter, we observed that RT improves intra-tumoral chemotherapy distribution. These effects were not hindered by anti-angiogenic sunitinib. Moreover, sub-optimal doses of doxorubicin had almost no effect alone, but significantly delayed tumor growth after RT. These data demonstrate that RT favors the efficacy of chemotherapy by improving tissue distribution, and could be an alternative chemosensitizing strategy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Hypoxia is a major risk factor of prostate cancer radioresistance. We evaluated hypoxia non-invasively, using 18 F-Misonidazole PET/CT prior to radiotherapy and after a dose of 20 Gy in intermediate-risk prostate cancer patients. Decreased hypoxic volumes were observed in all patients, suggesting that radiotherapy induces early prostate tumor reoxygenation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Hypoxia is a major factor in prostate cancer aggressiveness and radioresistance. Predicting which patients might be bad candidates for radiotherapy may help better personalize treatment decisions in intermediate-risk prostate cancer patients. We assessed spatial distribution of 18 F-Misonidazole (FMISO) PET/CT uptake in the prostate prior to radiotherapy treatment., Materials and Methods: Intermediate-risk prostate cancer patients about to receive high-dose (>74 Gy) radiotherapy to the prostate without hormonal treatment were prospectively recruited between 9/2012 and 10/2014. Prior to radiotherapy, all patients underwent a FMISO PET/CT as well as a MRI and 18 F-choline-PET. 18 F-choline and FMISO-positive volumes were semi-automatically determined using the fuzzy locally adaptive Bayesian (FLAB) method. In FMISO-positive patients, a dynamic analysis of early tumor uptake was performed. Group differences were assessed using the Wilcoxon signed rank test. Parameters were correlated using Spearman rank correlation., Results: Of 27 patients (median age 76) recruited to the study, 7 and 9 patients were considered positive at 2.5h and 3.5h FMISO PET/CT respectively. Median SUV max and SUV max tumor to muscle (T/M) ratio were respectively 3.4 and 3.6 at 2.5h, and 3.2 and 4.4 at 3.5h. The median FMISO-positive volume was 1.1 ml., Conclusions: This is the first study regarding hypoxia imaging using FMISO in prostate cancer showing that a small FMISO-positive volume was detected in one third of intermediate-risk prostate cancer patients., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

The concept of hypofractionation is gaining momentum in radiation oncology centres, enabled by recent advances in radiotherapy apparatus. The gain of efficacy of this innovative treatment must be defined. We present a computer model based on translational murine data for in silico testing and optimization of various radiotherapy protocols with respect to tumour resistance and the microenvironment heterogeneity. This model combines automata approaches with image processing algorithms to simulate the cellular response of tumours exposed to ionizing radiation, modelling the alteration of oxygen permeabilization in blood vessels against repeated doses, and introducing mitotic catastrophe (as opposed to arbitrary delayed cell-death) as a means of modelling radiation-induced cell death. Published data describing cell death in vitro as well as tumour oxygenation in vivo are used to inform parameters. Our model is validated by comparing simulations to in vivo data obtained from the radiation treatment of mice transplanted with human prostate tumours. We then predict the efficacy of untested hypofractionation protocols, hypothesizing that tumour control can be optimized by adjusting daily radiation dosage as a function of the degree of hypoxia in the tumour environment. Further biological refinement of this tool will permit the rapid development of more sophisticated strategies for radiotherapy.

Guidance molecules were first described in the nervous system to control axon outgrowth direction. They are also widely expressed outside the nervous system where they control cell migration, tissue development and establishment of the vascular network. In addition, they are involved in cancer development, tumor angiogenesis and metastasis. This review is primarily focused on their functions in lung cancer and their involvement in lung development is also presented. Five guidance molecule families and their corresponding receptors are described, including the semaphorins/neuropilins/plexins, ephrins and Eph receptors, netrin/DCC/UNC5, Slit/Robo and Notch/Delta. In addition, the possibility to target these molecules as a therapeutic approach in cancer is discussed.

New histone deacetylase inhibitors have been synthesized and evaluated for their activity against non-small lung cancer cell line H661. These compounds have been designed with diversely substituted 1,4-benzodiazepine-2,5-dione moieties as cyclic peptide mimic cap structures, and a hydroxamate side chain. Biological evaluations demonstrated that benzodiazepine-based HDACi bearing an aromatic substituent at the N1 position exhibited promising antiproliferative and HDAC-inhibitory activities.

Semaphorins, a family of genes encoding guidance molecules in the nervous system, influence a variety of cellular mechanisms including migration, proliferation and cytoskeleton reorganization. Interestingly, many members are expressed throughout lymphoid tissues and by different immune cells like lymphocytes, NK, monocytes and dendritic cells. Besides, the array of functions semaphorins usually regulate during organogenesis coincide with several key events required for the initiation as well as the regulation of the host immune response. Thus, it is not surprising if a substantial number of them modulates immune processes such as the establishment of the immunological synapse, differentiation to effector and helper cells, clonal expansion, migration and phagocytosis. For this purpose, immune semaphorins can signal via their canonical plexin receptors but also possibly by unique discrete cell surface proteins or associations thereof expressed by, and critical to, leukocytes. A growing list of semaphorins, receptors or related molecules keep being reported in the immune system, and display nonredundant roles at controlling its integrity and efficacy.

The tumor suppressor gene, Semaphorin SEMA3F, is frequently downregulated in lung cancer. Understanding the specific mechanism of SEMA3F suppression should be informative in terms of epithelial carcinogenesis and potential therapeutic interventions. Although a CpG-island is located 5083-3927 nt upstream of the translation start site, there have been no previous reports dealing with SEMA3F promoter regulation. We have now mapped the transcriptional initiation sites within the CpG-island and defined the region necessary for transcriptional activation. We then looked for evidence of SEMA3F promoter methylation since SEMA3F mutations are rare. By Southern blot and methylation-specific PCR assays, we identified a region in cell lines (i.e., area d at position minus 3850-3644 nt) for which methylation was significantly (P<0.0001) correlated with loss of expression. However, histone deacetylase inhibition with Trichostatin A was much more effective than 5-aza-2'-deoxycytidine in stimulating SEMA3F. Our results suggest that while SEMA3F promoter methylation correlates with repression, chromatin remodeling through histone deacetylase inhibition is sufficient to activate SEMA3F expression.

Semaphorins--a family of secreted, membrane-bound, and transmembrane proteins--play an important role in the development of various organs, as well as in axonal pathfinding, angiogenesis, tumorigenesis, and the immunological response. Neuropilins 1 and 2 (NRP1 and 2) are receptors for the class 3 secreted semaphorins (SEMA3s) but not for the other classes of semaphorins. NRPs are also coreceptors for vascular endothelial growth factor 165 (VEGF165), suggesting that SEMA3s could inhibit the VEGF165-VEGF receptor (VEGFR) pathway during angiogenesis. Until recently, it was believed that binding of SEMA3s to neuropilins was necessary to initiate signaling from plexins, the active players in semaphorin signal transduction. However, Gu and colleagues have recently described an exception: Their research suggests that SEMA3E signal transduction may be neuropilin independent. This Perspective focuses on this recent finding in the context of semaphorin signaling outside the nervous system.