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Purpose: Hypoxia in tumors is associated with increased malignancy and resistance to conventional photon radiation therapy. This study investigated the potential of particle therapy to counteract radioresistance in syngeneic rat prostate carcinoma., Methods and Materials: Subcutaneously transplanted R3327-HI tumors were irradiated with photons or carbon ions under acute hypoxic conditions, induced by clamping the tumor-supplying artery 10 min before and during irradiation. Dose-response curves were determined for the endpoint "local tumor control within 300 days" and compared with previously published data acquired under oxic conditions. Doses at 50% tumor control probability (TCD 50 ) were used to quantify hypoxia-induced radioresistance relative to that under oxic conditions and also to quantify the increased effectiveness of carbon ions under oxic and hypoxic conditions relative to photons., Results: Compared with those under oxic conditions, TCD 50 values under hypoxic conditions increased by a factor of 1.53 ± 0.08 for photons and by a factor of 1.28 ± 0.06 for carbon ions (oxygen enhancement ratio). Compared with those for photons, TCD 50 values for carbon ions decreased by a factor of 2.08 ± 0.13 under oxic conditions and by a factor of 2.49 ± 0.08 under hypoxic conditions (relative biological effectiveness). While the slope of the photon dose-response curves increased when changing from oxic to hypoxic conditions, the slopes were steeper and remained unchanged for carbon ions., Conclusions: The reduced oxygen enhancement ratio of carbon ions indicated that the required dose increase in hypoxic tumors was 17% lower for carbon ions than for photons. Additionally, carbon ions reduced the effect of intertumor heterogeneity on the radiation response. Therefore, carbon ions may confer a significant advantage for the treatment of hypoxic tumors that are highly resistant to conventional photon radiation therapy., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective . We propose a criterion of biological effectiveness of nonuniform hypoxia-targeted dose distributions in heterogeneous hypoxic tumors based on equivalent uniform aerobic dose (EUAD). We demonstrate the utility of this criterion by applying it to the model problems in radiotherapy for tumors with different levels of oxygen enhancement ratio (OER) and different degrees of dose nonuniformity. Approach . The EUAD is defined as the uniform dose that, under well-oxygenated conditions, produces equal integrated survival of clonogenic cells in radiotherapy for heterogeneous hypoxic tumors with a non-uniform dose distribution. We define the dose nonuniformity effectiveness (DNE) in heterogeneous tumors as the ratio of the EUAD( D N ) for a non-uniform distribution D N and the reference EUAD( D U ) for the uniform dose distribution D U with equal integral tumor dose. The DNE concept is illustrated in a radiotherapy model problem for non-small cell lung cancer treated with hypoxia targeted dose escalation. A two-level cell population tumor model was used to consider the hypoxic and oxygenated tumor cells. Results . Theoretical analysis of the DNE shows that the entire region of the OER can be separated in two regions by a threshold OER th : (1) OER > OER th where DNE > 1 indicating higher effectiveness of nonuniform dose distributions and (2) OER < OER th where DNE < 1 indicating higher effectiveness of uniform dose distributions. Our simulations show that the value of the threshold OER th in radiotherapy with conventional fractionation is significant in the range of about 1.2-1.6 depending on selected radiotherapy parameters. In general, the OER th increases with reoxygenation rate, relative hypoxic volume and dose escalation factor. The threshold value of OER th is smaller of about 1.1 for hypofractionated radiotherapy. Significance . The analysis of dose distributions using the DNE shows that the uniform dose distributions may improve biological cell killing effect in heterogeneous tumors with intermediate oxygen levels compared to targeted nonuniform dose distribution., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

A large proportion of cancer patients present with unresectable bulky disease at baseline or following treatment failure. The data available in the literature suggest that the vast majority of these patients do not benefit from available standard therapies. Therefore the clinical outcomes are poor; patients are desperate and usually relegated to palliative or best supportive care as the only options. Large tumor masses are usually hypoxic, resistant to radiation and systemic therapy, with extensive regional infiltration of the surrounding critical organs, the presence of which makes it impossible to deliver a radical dose of radiation. Promising data in terms of improved therapeutic ratio where such complex tumors are concerned can be seen with the use of new emerging unconventional radiotherapy techniques known as spatially fractionated radiotherapies (SFRT). One of them is PATHY, or PArtial Tumor irradiation targeting HYpoxic segment, which is characterized by a very short treatment course offering a large spectrum of therapeutic benefits in terms of the symptom relief, quality of life, local tumor control, neoadjuvant and immunomodulatory effects., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tumor hypoxia has been shown to predict poor patient outcomes in several cancer types, partially because it reduces radiation's ability to kill cells. We hypothesized that some of the clinical effects of hypoxia could also be due to its impact on the tumor microbiome. Therefore, we examined the RNA sequencing data from the Oncology Research Information Exchange Network database of patients with colorectal cancer treated with radiotherapy. We identified microbial RNAs for each tumor and related them to the hypoxic gene expression scores calculated from host mRNA. Our analysis showed that the hypoxia expression score predicted poor patient outcomes and identified tumors enriched with certain microbes such as Fusobacterium nucleatum. The presence of other microbes, such as Fusobacterium canifelinum, predicted poor patient outcomes, suggesting a potential interaction between hypoxia, the microbiome, and radiation response. To experimentally investigate this concept, we implanted CT26 colorectal cancer cells into immune-competent BALB/c and immune-deficient athymic nude mice. After growth, in which tumors passively acquired microbes from the gastrointestinal tract, we harvested tumors, extracted nucleic acids, and sequenced host and microbial RNAs. We stratified tumors based on their hypoxia score and performed a metatranscriptomic analysis of microbial gene expression. In addition to hypoxia-tropic and -phobic microbial populations, analysis of microbial gene expression at the strain level showed expression differences based on the hypoxia score. Thus, hypoxia gene expression scores seem to associate with different microbial populations and elicit an adaptive transcriptional response in intratumoral microbes, potentially influencing clinical outcomes., Significance: Tumor hypoxia reduces radiotherapy efficacy. In this study, we explored whether some of the clinical effects of hypoxia could be due to interaction with the tumor microbiome. Hypoxic gene expression scores associated with certain microbes and elicited an adaptive transcriptional response in others that could contribute to poor clinical outcomes., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: To address the problem of suboptimal reactive oxygen species (ROS) production in Radiation therapy (RT) which was resulted from exacerbated tumor hypoxia and the heterogeneous distribution of radiation sensitizers., Materials and Methods: In this work, a novel nanomedicine, designated as PLGA@IR780-Bi-DTPA (PIBD), was engineered by loading the radiation sensitizer Bi-DTPA and the photothermal agent IR780 onto poly(lactic-co-glycolic acid) (PLGA). This design leverages the tumor-targeting ability of IR780 to ensure selective accumulation of the nanoparticles in tumor cells, particularly within the mitochondria. The effect of the photothermal therapy-enhanced radiation therapy was also examined to assess the alleviation of hypoxia and the enhancement of radiation sensitivity., Results: The PIBD nanoparticles exhibited strong capacity in mitochondrial targeting and selective tumor accumulation. Upon activation by 808 nm laser irradiation, the nanoparticles effectively alleviated local hypoxia by photothermal effect enhanced blood supplying to improve oxygen content, thereby enhancing the ROS production for effective RT. Comparative studies revealed that PIBD-induced RT significantly outperformed conventional RT in treating hypoxic tumors., Conclusion: This design of tumor-targeting photothermal therapy-enhanced radiation therapy nanomedicine would advance the development of targeted drug delivery system for effective RT regardless of hypoxic microenvironment., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Zhang et al.)

Hypoxia (oxygen deprivation) occurs in most solid malignancies, albeit with considerable heterogeneity. Hypoxia is associated with an aggressive cancer phenotype by promotion of genomic instability, evasion of anti-cancer therapies including radiotherapy and enhancement of metastatic risk. Therefore, hypoxia results in poor cancer outcomes. Targeting hypoxia to improve cancer outcomes is an attractive therapeutic strategy. Hypoxia-targeted dose painting escalates radiotherapy dose to hypoxic sub-volumes, as quantified and spatially mapped using hypoxia imaging. This therapeutic approach could overcome hypoxia-induced radioresistance and improve patient outcomes without the need for hypoxia-targeted drugs. This article will review the premise and underpinning evidence for personalized hypoxia-targeted dose painting. It will present data on relevant hypoxia imaging biomarkers, highlight the challenges and potential benefit of this approach and provide recommendations for future research priorities in this field. Personalized hypoxia-based radiotherapy de-escalation strategies will also be addressed., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background and Purpose: In this study, fluoromisonidazole positron emission tomography (F-MISO PET/CT) was used to evaluate tumor hypoxia and re-oxygenation in patients with lung tumors treated with stereotactic body radiation therapy (SBRT)., Materials and Methods: Patients with T1-2 N0 lung cancer were included in this study. The prescribed dose was 48-52 Gy in four fractions. F-MISO PET/CT was performed twice, before SBRT and 1-3 days after the first irradiation. The maximum standardized uptake value (SUVmax) and tumor/muscle ratio (TMR) were evaluated as indicators of hypoxia. The threshold for hypoxia was defined as a TMR of 1.30 or more., Results: Between 2016 and 2021, 15 patients were included. Pre-treatment tumor hypoxia was observed in nine tumors (60 %). TMR in all six tumors without pre-treatment hypoxia rose after single high-dose irradiation. In contrast, TMR in six of nine tumors with pre-treatment hypoxia dropped after irradiation, suggesting re-oxygenation. Although no local recurrence was noted, regional and/or distant relapses were seen in four patients (27 %). Of these, three had tumors with abnormal F-MISO uptake. The remaining patient had a tumor without signs of hypoxia on pre-treatment PET/CT. The 2-year progression free survival of patients with tumors with and without pre-treatment hypoxia were 30 % and 63 %, respectively (p = 0.319)., Conclusion: Tumor hypoxia reduced after single high-dose irradiation. Tumor with F-MISO uptake seems to be an unfavorable prognostic factor in lung SBRT., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Radiotherapy is a crucial cancer treatment, but its outcome is still far from satisfactory. One of the reasons that cancer cells show resistance to ionizing radiation is hypoxia, defined as a low level of oxygenation, which is typical for solid tumors. In the hypoxic environment, cancer cells are 2-3 times more resistant to ionizing radiation than normoxic cells. To overcome this important impediment, radiosensitizers should be introduced to cancer therapy. When modified with an electrophilic substituent, nucleosides may undergo efficient dissociative electron attachment (DEA) that leaves behind nucleoside radicals, which, in secondary reactions, are able to induce DNA damage, leading to cancer cell death. We report the radiosensitizing effect of one of the best-known DEA-type radiosensitizers-5-bromo-2'-deoxyuridine (BrdU)-on breast (MCF-7) and prostate (PC3) cancer cells under both normoxia and hypoxia. MCF-7 and PC3 cells were treated with BrdU to investigate the effect of hypoxia on cell proliferation, incorporation into DNA and radiosensitivity. While the oxygen concentration did not significantly affect the efficiency of BrdU incorporation into DNA or the proliferation of tumor cells, the radiosensitizing effect of BrdU on hypoxic cells was more evident than on normoxic cells. Further mechanistic studies performed with the use of flow cytometry showed that under hypoxia, BrdU increased the level of histone H2A.X phosphorylation after X-ray exposure to a greater extent than under normal oxygenation conditions. These results confirm that the formation of double-strand breaks in hypoxic BrdU-treated cancer cells is more efficient. In addition, by performing stationary radiolysis of BrdU solution in the presence of an ● OH radical scavenger, we compared the degree of its electron-induced degradation under aerobic and anaerobic conditions. It was determined that radiodegradation under anaerobic conditions was almost twice as high as that under aerobic conditions.

The receptor tyrosine kinase AXL (RTK-AXL) is implicated in therapy resistance and tumor progression in glioblastoma multiforme (GBM). Here, we investigated therapy-induced receptor modifications and how endogenous RTK-AXL expression and RTK-AXL inhibition contribute to therapy resistance in GBM. GBM cell lines U118MG and SF126 were exposed to temozolomide (TMZ) and radiation (RTX). Receptor modifications in response to therapy were investigated on protein and mRNA levels. TMZ-resistant and RTK-AXL overexpressing cell lines were exposed to increasing doses of TMZ and RTX, with and without RTK-AXL tyrosine kinase inhibitor (TKI). Colorimetric microtiter (MTT) assay and colony formation assay (CFA) were used to assess cell viability. Results showed that the RTK-AXL shedding product, C-terminal AXL (CT-AXL), rises in response to repeated TMZ doses and under hypoxia, acts as a surrogate marker for radio-resistance. Endogenous RTX-AXL overexpression leads to therapy resistance, whereas combination therapy of TZM and RTX with TKI R428 significantly increases therapeutic effects. This data proves the role of RTK-AXL in acquired and intrinsic therapy resistance. By demonstrating that therapy resistance may be overcome by combining AXL TKI with standard treatments, we have provided a rationale for future study designs investigating AXL TKIs in GBM.

Anticancer treatment is largely affected by the hypoxic tumor microenvironment (TME), which causes the resistance of the tumor to radiotherapy. Combining radiosensitizer compounds and O 2 self-enriched moieties is an emerging strategy in hypoxic-tumor treatments. Herein, we engineered GdW 10 @PDA-CAT (K 3 Na 4 H 2 GdW 10 O 36 ·2H 2 O, GdW 10 , polydopamine, PDA, catalase, CAT) composites as a radiosensitizer for the TME-manipulated enhancement of radiotherapy. In the composites, Gd (Z = 64) and W (Z = 74), as the high Z elements, make X-ray gather in tumor cells, thereby enhancing DNA damage induced by radiation. CAT can convert H 2 O 2 to O 2 and H 2 O to enhance the X-ray effect under hypoxic TME. CAT and PDA modification enhances the biocompatibility of the composites. Our results showed that GdW 10 @PDA-CAT composites increased the efficiency of radiotherapy in HT29 cells in culture. This polyoxometalates and O 2 self-supplement composites provide a promising radiosensitizer for the radiotherapy field.

Background: Hypoxia is known to be prevalent in solid tumors such as non-small cell lung cancer (NSCLC) and reportedly correlates with poor prognostic clinical outcome. PET imaging can provide in-vivo hypoxia measurements to support targeted radiotherapy treatment planning. We explore the potential of proton therapy in performing patient-specific dose escalation and compare it with photon volumetric modulated arc therapy (VMAT)., Methods: Dose escalation has been calibrated to the patient specific tumor response of ten stage IIb-IIIb NSCLC patients by combining HX4-PET imaging and radiobiological modelling of oxygen enhancement ratio (OER) to target variable tumor hypoxia. In a dose-escalation-by-contour approach, escalated dose levels were simulated to the most hypoxic region of the primary target and its effectiveness in improving loco-regional tumor control was assessed. Furthermore, the impact on normal tissue of proton treatments including dose escalation was evaluated in comparison to the normal tissue complication probability (NTCP) of conventional VMAT plans., Results: Ignoring regions of tumor hypoxia can cause overestimation of TCP values by up to 10%, which can effectively be recovered on average to within 0.9% of the nominal TCP, using patient-specific dose escalations of up to 22% of the prescribed dose to PET defined hypoxic regions. Despite such dose escalations, the use of protons could also simultaneously reduce mean doses to the heart (- 14.3 Gy RBE ), lung (- 8.3 Gy RBE ), esophagus (- 6.9 Gy RBE ) and spinal cord (- 3.8 Gy) compared to non-escalated VMAT plans. These reductions are predicted to lead to clinically relevant decreases in NTCP for radiation-induced pneumonitis (- 11.3%), high grade heart toxicity (- 7.4%) and esophagitis (- 7.5%)., Conclusions: This study suggests that the administration of proton therapy for dose escalation to patient specific regions of tumor hypoxia in the treatment of NSCLC can mitigate TCP reduction due to hypoxia-induced radio resistance, while simultaneously reducing NTCP levels even when compared to non-escalated treatments delivered with state-of-the-art photon techniques., (© 2021. The Author(s).)

Purpose: The differential response of normal and tumor tissues to ultrahigh-dose-rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study was to investigate these effects within hypoxic multicellular tumor spheroids through simulations and experiments., Methods and Materials: Physicobiological equations were derived to model (1) the diffusion and metabolism of oxygen within spheroids; (2) its depletion through reactions involving radiation-induced radicals; and (3) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored postradiation. Clonogenic survival of 2 other cell lines was also investigated., Results: The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroids compared with conventional irradiation, but no significant difference was observed for well-oxygenated 2-dimensional cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy., Conclusions: Tumor spheroids can be used as a model to study FLASH irradiation in vitro. The improved survival of tumor spheroids receiving FLASH radiation confirms that ultrafast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: To review the radiobiological mechanisms of stereotactic body radiation therapy stereotactic body radiation therapy (SBRT) and stereotactic radiation surgery (SRS)., Methods and Materials: We reviewed previous reports and recent observations on the effects of high-dose irradiation on tumor cell survival, tumor vasculature, and antitumor immunity. We then assessed the potential implications of these biological changes associated with SBRT and SRS., Results: Irradiation with doses higher than approximately 10 Gy/fraction causes significant vascular injury in tumors, leading to secondary tumor cell death. Irradiation of tumors with high doses has also been reported to increase the antitumor immunity, and various approaches are being investigated to further elevate antitumor immunity. The mechanism of normal tissue damage by high-dose irradiation needs to be further investigated., Conclusions: In addition to directly killing tumor cells, high-dose irradiation used in SBRT and SRS induces indirect tumor cell death via vascular damage and antitumor immunity. Further studies are warranted to better understand the biological mechanisms underlying the high efficacy of clinical SBRT and SRS and to further improve the efficacy of SBRT and SRS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tumor hypoxia in head-and-neck squamous cell carcinoma (HNSCC) leads to an immunosuppressive microenvironment and reduces the response to radiotherapy. In this prospective imaging trial, we investigated potential interactions between functional hypoxia imaging and infiltrating lymphocyte levels as a potential predictor for treatment response in HNSCC patients. Methods: In total, 49 patients receiving definitive chemoradiation for locally advanced HNSCCs underwent pretherapeutic biopsies and peritherapeutic hypoxia imaging using 18 F-misonidazole PET at weeks 0, 2, and 5 during chemoradiation. Hematoxylin-eosin and immunohistochemical stainings for tumor-infiltrating lymphocytes, tissue-based hypoxia, and microvascular markers were analyzed and correlated with the longitudinal hypoxia dynamics and patient outcomes. Results: High levels of tumor-infiltrating total lymphocytes correlated with superior locoregional control (LRC) (hazard ratio [HR], 0.279; P = 0.011) and progression-free survival (PFS) (HR, 0.276; P = 0.006). Similarly, early resolution of 18 F-misonidazole PET-detected tumor hypoxia quantified by 18 F-misonidazole dynamics between weeks 0 and 2 of chemoradiation was associated with improved LRC (HR, 0.321; P = 0.015) and PFS (HR, 0.402; P = 0.043). Outcomes in the favorable early hypoxia resolution subgroup significantly depended on infiltrating lymphocyte counts, with patients who showed both an early hypoxia response and high lymphocyte infiltration levels exhibiting significantly improved LRC (HR, 0.259; P = 0.036) and PFS (HR, 0.242; P = 0.017) compared with patients with an early hypoxia response but low lymphocyte counts. These patients exhibited oncologic results comparable to those of patients with no hypoxia response within the first 2 wk of chemoradiation. Conclusion: This analysis established a clinical hypoxia-immune score that predicted treatment responses and outcomes in HNSCC patients undergoing chemoradiation and may help to devise novel concepts for biology-driven personalization of chemoradiation., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: The extreme microscopic heterogeneity of tumors makes it difficult to characterize tumor hypoxia. We evaluated how changes in the spatial resolution of oxygen imaging could alter measures of tumor hypoxia and their correlation to radiation therapy response., Methods and Materials: Cherenkov-Excited Luminescence Imaging in combination with an oxygen probe, Oxyphor PtG4 was used to directly image tumor pO 2 distributions with 0.2 mm spatial resolution at the time of radiation delivery. These pO 2 images were analyzed with variations of reduced spatial resolution from 0.2 mm to 5 mm, to investigate the influence of how reduced imaging spatial resolution would affect the observed tumor hypoxia. As an in vivo validation test, mice bearing tumor xenografts were imaged for hypoxic fraction and median pO 2 to examine the predictive link with tumor response to radiation therapy, while accounting for spatial resolution., Results: In transitioning from voxel sizes of 200 μm to 3 mm, the median pO 2 values increased by a few mm Hg, and the hypoxic fraction decreased by more than 50%. When looking at radiation-responsive tumors, the median pO 2 values changed just a few mm Hg as a result of treatment, and the hypoxic fractions changed by as much as 50%. This latter change, however, could only be seen when sampling was performed with high spatial resolution. Median pO 2 or similar quantities obtained from low resolution measurements are commonly used in clinical practice, however these parameters are much less sensitive to changes in the tumor microenvironment than the tumor hypoxic fraction obtained from high-resolution oxygen images., Conclusions: This study supports the hypothesis that for adequate measurements of the tumor response to radiation therapy, oxygen imaging with high spatial resolution is required to accurately characterize the hypoxic fraction., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The outcome of radiotherapy is significantly restricted by tumor hypoxia. To overcome this obstacle, one prevalent solution is to increase intratumoral oxygen supply. However, its effectiveness is often limited by the high metabolic demand for O 2 by cancer cells. Herein, we develop a hybrid semiconducting organosilica-based O 2 nanoeconomizer pHPFON-NO/O 2 to combat tumor hypoxia. Our solution is twofold: first, the pHPFON-NO/O 2 interacts with the acidic tumor microenvironment to release NO for endogenous O 2 conservation; second, it releases O 2 in response to mild photothermal effect to enable exogenous O 2 infusion. Additionally, the photothermal effect can be increased to eradicate tumor residues with radioresistant properties due to other factors. This "reducing expenditure of O 2 and broadening sources" strategy significantly alleviates tumor hypoxia in multiple ways, greatly enhances the efficacy of radiotherapy both in vitro and in vivo, and demonstrates the synergy between on-demand temperature-controlled photothermal and oxygen-elevated radiotherapy for complete tumor response.

Electrochemotherapy (ECT) is a new and promising treatment strategy for cancer treatment. The aim of this work is to investigate the effect of 900 MHz radiofrequency electromagnetic fields (RF-EMFs) on the mechanisms of ECT (low voltage, high frequency) including cell permeability in vitro, and tumor hypoxia, immune system response in vivo, and on volume of tumors treated with ECT (70 V/cm, 5 kHz). The 4T1 cells were exposed to RF-EMFs at 17, 162, or 349 µW/cm 2 power densities, using GSM900 simulator, 10 min. The cells were then put in individual groups, comprising of no treatment, chemotherapy, electric pulses (EPs), or ECT. The cell viability was evaluated. The mice with 4T1 tumor cells were exposed to RF field 10 min/day until the tumor volume reached about 8 mm. Then, the mice tumors were treated with ECT. Tumor hypoxia and immune system response was analyzed through immunohistochemistry (IHC) assay and ELISA technique, respectively. The volume of tumors was also calculated for 24 days following the treatment. The results showed that RF fields at 349 µW/cm 2 could increase tumor hypoxia induced by ECT and cause a significant increase of Interferon-gamma (IFN-γ) in comparison with group ECT alone. However, 900 MHz radiations did not affect the volume of tumors treated to ECT (70 V/cm, 5 kHz) significantly. In this study, 900 MHz EMF could improve some biological pathways induced by ECT. Such a positive effect could utilize in some other treatments to increase efficacy, which should be investigated in further research.

Solid tumors inevitably develop radioresistance due to low oxygen partial pressure in the tumor microenvironment. Despite numerous attempts, there are still few effective ways to avoid the hypoxia-induced poor radiotherapeutic effect. To overcome this problem, platinum (Pt) nanodots were fabricated into a mesoporous bismuth (Bi)-based nanomaterial to construct a biodegradable nanocomposite BiPt-folic acid-modified amphiphilic polyethylene glycol (PFA). BiPt-PFA could act as a radiosensitizer to enhance the absorption of X-rays at the tumor site and simultaneously trigger response behaviors related to the tumor microenvironment due to the enrichment of materials in the tumor area. During this process, the Bi-based component consumed glutathione via coordination, thus altering the oxidative stress balance, while Pt nanoparticles catalyzed the decomposition of hydrogen peroxide to generate oxygen, thereby relieving tumor hypoxia. Both Pt and Bi thus co-modulated the tumor microenvironment to improve the radiotherapeutic effect. In addition, Pt dots in BiPt-PFA had strong near-infrared absorption ability and created an intensive photothermal therapeutic effect. Modulation of the tumor microenvironment could thus improve the therapeutic effect in hypoxic tumors by a combination of photothermal therapy and enhanced radiotherapy. BiPt-PFA, as a biodegradable nanocomposite, may thus modulate the tumor microenvironment to enhance the hypoxic tumor therapeutic effect by thermoradiotherapy.

Esophageal squamous cell carcinoma (ESCC), the most frequent esophageal cancer (EC) subtype, entails dismal prognosis. Hypoxia, a common feature of advanced ESCC, is involved in resistance to radiotherapy (RT). RT response in hypoxia might be modulated through epigenetic mechanisms, constituting novel targets to improve patient outcome. Post-translational methylation in histone can be partially modulated by histone lysine demethylases (KDMs), which specifically removes methyl groups in certain lysine residues. KDMs deregulation was associated with tumor aggressiveness and therapy failure. Thus, we sought to unveil the role of Jumonji C domain histone lysine demethylases (JmjC-KDMs) in ESCC radioresistance acquisition. The effectiveness of RT upon ESCC cells under hypoxic conditions was assessed by colony formation assay. KDM3A/KDM6B expression, and respective H3K9me2 and H3K27me3 target marks, were evaluated by RT-qPCR, Western blot, and immunofluorescence. Effect of JmjC-KDM inhibitor IOX1, as well as KDM3A knockdown, in in vitro functional cell behavior and RT response was assessed in ESCC under hypoxic conditions. In vivo effect of combined IOX1 and ionizing radiation treatment was evaluated in ESCC cells using CAM assay. KDM3A, KDM6B, HIF-1α, and CAIX immunoexpression was assessed in primary ESCC and normal esophagus. Herein, we found that hypoxia promoted ESCC radioresistance through increased KDM3A/KDM6B expression, enhancing cell survival and migration and decreasing DNA damage and apoptosis, in vitro. Exposure to IOX1 reverted these features, increasing ESCC radiosensitivity and decreasing ESCC microtumors size, in vivo. KDM3A was upregulated in ESCC tissues compared to the normal esophagus, associating and colocalizing with hypoxic markers (HIF-1α and CAIX). Therefore, KDM3A upregulation in ESCC cell lines and primary tumors associated with hypoxia, playing a critical role in EC aggressiveness and radioresistance. KDM3A targeting, concomitant with conventional RT, constitutes a promising strategy to improve ESCC patients' survival.

As a common feature in a majority of malignant tumors, hypoxia has become the Achilles' heel of photodynamic therapy (PDT). The development of type-I photosensitizers that show hypoxia-tolerant PDT efficiency provides a straightforward way to address this issue. However, type-I PDT materials have rarely been discovered. Herein, a π-conjugated molecule with A-D-A configuration, COi6-4Cl, is reported. The H 2 O-dispersible nanoparticle of COi6-4Cl can be activated by an 880 nm laser, and displays hypoxia-tolerant type I/II combined PDT capability, and more notably, a high NIR-II fluorescence with a quantum yield over 5%. Moreover, COi6-4Cl shows a negligible photothermal conversion effect. The non-radiative decay of COi6-4Cl is suppressed in the dispersed and aggregated state due to the restricted molecular vibrations and distinct intermolecular steric hindrance induced by its four bulky side chains. These features make COi6-4Cl a distinguished single-NIR-wavelength-activated phototheranostic material, which performs well in NIR-II fluorescence-guided PDT treatment and shows an enhanced in vivo anti-tumor efficiency over the clinically approved Chlorin e6, by the equal stresses on hypoxia-tolerant anti-tumor therapy and deep-penetration imaging. Therefore, the great potential of COi6-4Cl in precise PDT cancer therapy against hypoxia challenges is demonstrated., (© 2020 Wiley-VCH GmbH.)

A family of five heteroleptic complexes [Ru(C^N)(N^N) 2 ][PF 6 ] (HC^N = methyl 1-butyl-2-arylbenzimidazolecarboxylate; N^N = polypyridine) has been synthesized to act as biologically-compatible green light photosensitizers (PSs) with phototherapeutic indexes (PIs) up to higher than 700 under hypoxia (2% O 2 ) in HeLa cancer cells under short time of irradiation.

Objective: As a fractionated course of radiotherapy proceeds tumour shrinkage leads to resolution of hypoxia and the initiation of accelerated proliferation of radioresistant cancer cells with better repair capacity. We hypothesise that, in tumours with significant hypoxia, improved tumour control could be achieved with biphasic fractionation schedules that either use acceleration after 3-4 weeks of conventional radiotherapy or deliver a higher proportional dose towards the end of a course of treatment. We conducted a modelling study based on the concept of biological effective dose (BED) comparing such novel regimens with conventional fractionation., Methods: The comparator conventional fractionation schedule 70 Gy in 35 fractions delivered over 7 weeks was tested against the following novel regimens, both of which were designed to be isoeffective in terms of late normal tissue toxicity.40 Gy in 20 fractions over 4 weeks followed by 22.32 Gy in 6 consecutive daily fractions (delayed acceleration)30.4 Gy in 27 fractions over 4 weeks followed by 40 Gy in 15 fractions over 3 weeks (temporal dose redistribution)The delayed acceleration regimen is exactly identical to that of the comparator schedule over the first 28 days and the BED gains with the novel schedule are achieved during the second phase of treatment when reoxygenation is complete. For the temporal redistribution regimen, it was assumed that the reoxygenation fraction progressively increases during the first 4 weeks of treatment and an iterative approach was used to calculate the final tumour BED for varying hypoxic fractions., Results: Novel fractionation with delayed acceleration or temporal fractionation results in tumour BED gains equivalent to 3.5-8 Gy when delivered in 2 Gy fractions., Conclusion: In hypoxic tumours, novel fractionation strategies result in significantly higher tumour BED in comparison to conventional fractionation., Advances in Knowledge: We demonstrate that novel biphasic fractionation regimens could overcome the effects of tumour hypoxia resulting in biological dose escalation.

Purpose: Radiotherapy (RT) is one of the major treatments of cervical cancer. Although the prognosis of clinical cervical cancer becomes better in recent years, some patients still suffer from the recurrence and metastasis. Insufficiency of glucose and oxygen supply could increase the radioresistance of cervical cancer cells through regulating hypoxia-inducible factor 1 (HIF-1) in tumor microenvironment and glucose metabolism. And, berberine can regulate HIF-1. However, how berberine regulates tumor microenvironment and radioresistance through HIF-1 remains to be elucidated. Materials and methods: The human HeLa cervical cancer cells were treated with berberine and radiation under the high and low concentrations of glucose and oxygen, respectively. The survival of cells was tested by CCK-8 assay and colony formation assay. We investigated the PI3K- and IDH3α-related pathway molecules that may regulate HIF-1α by qPCR and western blot. Differentially expressed genes (DEGs) were identified by integrating five related cohort profile datasets. Protein-protein interaction (PPI) network analyses of DEGs related to HIF-1α were conducted by using the STRING database and Cytoscape software. Results: Berberine dramatically damaged HeLa cells under hypoxic and low-glucose conditions compared with the normoxic and high-glucose conditions. The clonogenic assay indicated that the application of berberine decreased the number of colony counts compared to the negative control. Low doses of berberine might decrease the level of phospho-PI3K and HIF-1α under the nutrient-deprived conditions. Moreover, we found that most of the differentially expressed genes which were related to CDKN1B were the downstream molecules regulated by HIF-1α. Conclusion: The results indicated that berberine could dramatically overcome the low-glucose and hypoxia-induced radioresistance. And the regulation berberine on nutrition-deficient conditions might involve in PI3K/HIF-1 pathway. Thus, the interference of glucose metabolism by berberine might be an attractive method to eliminate radioresistant cells and improve radiotherapy efficacy.

We have previously demonstrated that endothelial targeting of gold nanoparticles followed by external beam irradiation can cause specific tumor vascular disruption in mouse models of cancer. The induced vascular damage may lead to changes in tumor physiology, including tumor hypoxia, thereby compromising future therapeutic interventions. In this study, we investigate the dynamic changes in tumor hypoxia mediated by targeted gold nanoparticles and clinical radiation therapy (RT). By using noninvasive whole-body fluorescence imaging, tumor hypoxia was measured at baseline, on day 2 and day 13, post-tumor vascular disruption. A 2.5-fold increase (P<0.05) in tumor hypoxia was measured two days after combined therapy, resolving by day 13. In addition, the combination of vascular-targeted gold nanoparticles and radiation therapy resulted in a significant (P<0.05) suppression of tumor growth. This is the first study to demonstrate the tumor hypoxic physiological response and recovery after delivery of vascular-targeted gold nanoparticles followed by clinical radiation therapy in a human non-small cell lung cancer athymic Foxn1nu mouse model., Competing Interests: The authors have declared that no competing interests exist.

Numerous studies have shown that exosomes play important roles in tumor biology development. However, the function of exosomal protein in cancer progression under different oxygen condition after irradiation is poorly understood. In this study, non-small cell lung cancer (NSCLC) A549 cells were γ-ray irradiated under normoxic or hypoxic conditions, then the exosomes released from the irradiated cells were collected and co-cultured with nonirradiated A549 cells or human umbilical vein endothelial cells (HUVECs). It was found that the exosomes significantly promoted the proliferation, migration and invasion of A549 cells as well as the proliferation and angiogenesis of HUVECs. Moreover, the exosomes released from hypoxic cells and/or irradiated cells had more powerful driving force in tumor progression compared to that generated from normoxia cells. Meanwhile, the proteins contained in the exosomes derived from A549 cells under different conditions were detected using tandem mass tag (TMT), and their expression profiles were analyzed. It was found that the exosome-derived protein of angiopoietin-like 4 (ANGPTL4) contributed to the migration of A549 cells as well as the angiogenesis of HUVECs, suggesting its potential as an effective diagnostic biomarker of metastasis and even a therapeutic target of lung cancer., (©2020 by Radiation Research Society. All rights of reproduction in any form reserved.)

Electrochemotherapy (ECT), the combination of electric pulses (EPs) and an anticancer drug, is a type of cancer treatment method. We investigated the effect of 217-Hz magnetic fields (MFs) similar to that generated by GSM900 mobile phones, as intervening factors, on proposed mechanisms of ECT including permeability, tumor hypoxia and immune system response. The 4T1 cells were exposed to extremely low-frequency (ELF)-MFs at 93, 120 or 159 µT intensities, generated by Helmholtz coils 10 min, and then put in individual groups, comprising no treatment, chemotherapy, EPs or ECT. The cell viability was evaluated. Then, two treatment protocols were selected for in vivo experiments. The mice with 4T1 tumor cells were exposed to ELF-MFs 10 min/day until the day their tumors reached 8 mm in diameter. Then, the tumors were treated to ECT. Tumor hypoxia and immune system response were analyzed through immunohistochemistry assay and enzyme-linked immunosorbent assay technique, respectively. The results in vitro indicated a significant decreased ECT efficacy of 60 V/cm, 5 kHz at the flux density of 93 µT. The results in vivo showed that pre-exposure to ELF-MFs could increase tumor hypoxia induced by ECT. In addition, exposure to ELF-MFs before ECT caused a significant increase in interferon-γ/interleukin-4 in comparison with ECT alone. More studies, including studies on the effect of ELF-MFs emitted from mobile phones on tumor volume changes induced by ECT, are needed to elucidate how the process of ECT is influenced by the MFs.

Background: Emerging biomarkers from medical imaging or molecular characterization of tumour biopsies open up for combining the two and exploiting their synergy in treatment planning of cancer patients. We generated a paired data set of imaging- and gene-based hypoxia biomarkers in cervical cancer, appraised the influence of intratumour heterogeneity in patient classification, and investigated the benefit of combining the methodologies in prediction of chemoradiotherapy failure., Methods: Hypoxic fraction from dynamic contrast enhanced (DCE)-MR images and an expression signature of six hypoxia-responsive genes were assessed as imaging- and gene-based biomarker, respectively in 118 patients., Findings: Dichotomous biomarker cutoff to yield similar hypoxia status by imaging and genes was defined in 41 patients, and the association was validated in the remaining 77 patients. The two biomarkers classified 75% of 118 patients with the same hypoxia status, and inconsistent classification was not related to imaging-defined intratumour heterogeneity in hypoxia. Gene-based hypoxia was independent on tumour cell fraction in the biopsies and showed minor heterogeneity across multiple samples in 9 tumours. Combining imaging- and gene-based classification gave a significantly better prediction of PFS than one biomarker alone. A combined dichotomous biomarker optimized in 77 patients showed a large separation in PFS between more and less hypoxic tumours, and separated the remaining 41 patients with different PFS. The combined biomarker showed prognostic value together with tumour stage in multivariate analysis., Interpretation: Combining imaging- and gene-based biomarkers may enable more precise and informative assessment of hypoxia-related chemoradiotherapy resistance in cervical cancer., Funding: Norwegian Cancer Society, South-Eastern Norway Regional Health Authority, and Norwegian Research Council., Competing Interests: Declaration of Competing Interest HL is registered as inventor of a patent application covering the clinical use of the hypoxia gene signature (WO2013/124,738)., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Tumor hypoxia, acidosis, and excessive reactive oxygen species (ROS) were the main characteristics of the bladder tumor microenvironment (TME), and abnormal TME led to autophagy activation, which facilitated cancer cell proliferation. The therapeutic efficacy of autophagy inhibitors might also be impeded by abnormal TME. To address these issues, we proposed a new strategy that utilized manganese dioxide (MnO 2 ) nanoparticles to optimize the abnormal TME and revitalize autophagy inhibitors, and both oxygenation and autophagy inhibition may sensitize the tumor cells to radiation therapy. Methods: By taking advantage of the strong affinity between negatively charged MnO 2 and positively charged chloroquine (CQ), the nanoparticles were fabricated by integrating MnO 2 and CQ in human serum albumin (HSA)-based nanoplatform (HSA-MnO 2 -CQ NPs). Results: HSA-MnO 2 -CQ NPs NPs efficiently generated O 2 and increased pH in vitro after reaction with H + /H 2 O 2 and then released the encapsulated CQ in a H + /H 2 O 2 concentration-dependent manner. The NPs restored the autophagy-inhibiting activity of chloroquine in acidic conditions by increasing its intracellular uptake, and markedly blocked hypoxia-induced autophagic flux. In vivo studies showed the NPs improved pharmacokinetic behavior of chloroquine and effectively accumulated in tumor tissues. The NPs exhibited significantly decreased tumor hypoxia areas and increased tumor pH, and had remarkable autophagy inhibition efficacy on bladder tumors. Finally, a significant anti-tumor effect achieved by the enhanced autophagy inhibition and radiation sensitization. Conclusions: HSA-MnO 2 -CQ NPs synergistically regulated the abnormal TME and inhibited autophagic flux, and effectively sensitized radiation therapy to treat bladder cancers., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Continuous irradiation during photodynamic therapy (PDT) inevitably induces tumor hypoxia, thereby weakening the PDT effect. In PDT-induced hypoxia, providing singlet oxygen from stored chemical energy may enhance the cell-killing effect and boost the therapeutic effect. Herein, we present a phototheranostic (DPPTPE@PEG-Py NPs) prepared by using a 2-pyridone-based diblock polymer (PEG-Py) to encapsulate a semiconducting, heavy-atom-free pyrrolopyrrolidone-tetraphenylethylene (DPPTPE) with high singlet-oxygen-generation ability both in dichloromethane and water. The PEG-Py can trap the 1 O 2 generated from DPPTPE under laser irradiation and form a stable intermediate of endoperoxide, which can then release 1 O 2 in the dark, hypoxic tumor microenvironment. Furthermore, fluorescence-imaging-guided phototherapy demonstrates that this phototheranostic could completely inhibit tumor growth with the help of laser irradiation., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: Most solid tumors contain areas of chronic hypoxia. Gold nanoparticles (GNP) have been extensively explored as enhancers of external beam radiation; however, GNP have lower cellular uptake in hypoxic conditions than under normoxic conditions. Conversely, the chelator diacetyl-bis (N(4)-methylthiosemicarbazonato) copper II (CuATSM) deposits copper in hypoxic regions, allowing for dose enhancement in previously inaccessible regions., Methods: External beam sources with different spectra were modeled using a Monte Carlo code (EGSnrc) to evaluate radioenhancement in a layered model with metal solutions. Also considered was a simple concentric layered tumor model containing a hypoxic core with each layer varying in concentrations of either copper or gold according to hypoxic conditions. Low energy external photon beams were then projected onto the tumor to determine the regional dose enhancement dependent on hypoxic conditions., Results: Dose enhancement was more pronounced for beam spectra with low energy photons (225 kVp) and was highly dependent on metal concentrations from 0.1 g/kg to 100 g/kg. Increasing the depth of the metallic solution layer from 1 cm to 6 cm decreased dose enhancement. A small increase in the dose enhancement factor (DEF) of 1.01 was predicted in the hypoxic regions of the tumor model with commonly used diagnostic concentrations of CuATSM. At threshold concentrations of toxic subcutaneous injection levels, the DEF increases to 1.02, and in simulation of a high concentration of CuATSM, the DEF increased to 1.07. High concentration treatments are also considered, as well as synergistic combinations of GNP/CuATSM treatments., Conclusion: The research presented is novel utilization of CuATSM to target hypoxic regions and act as a radiosensitizer by the nature of its ability to deposit copper metal in reduced tissue. We demonstrate CuATSM at high concentrations with low energy photons can increase dose deposition in hypoxic tumor regions., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Martinez et al.)

Reactive oxygen species (ROS)-mediated cell apoptosis has been a significant strategy for tumor oxidative damage, while tumor hypoxia is a major bottleneck for efficiency. Here, a novel TiO-porphyrin nanosystem (FA-TiOPs) is designed by encapsulating TiO-porphyrin (TiOP) in folate-liposome. The nanosysytem can photocatalyze H 2 O and tumor-overexpressed H 2 O 2, in situ generating sufficient ROS. TiOP can photosplit water to produce ·OH radical, H 2 O 2 , and O 2 . Generated O 2 not only conquers the hypoxia of tumor environment but also can be further excited by TiOP to 1 O 2 for killing tumor cells. Density functional theory calculations indicate that high energy in excited state (S 1 ) of TiOP and narrow gap energy between S 1 and the triplet excited state (T n ) might contribute to the efficient photocatalytic action. Moreover, the generated and overexpressed H 2 O 2 in tumors can also be photocatalyzed to generate 1 O 2 especially in acid condition, helpful to specific anticancer effect while harmless to normal tissues. This research might pave a new way to bypass the hypoxia-triggered problem for cancer therapy.

Photodynamic therapy (PDT) and radiotherapy (RT) are oxygen-dependent treatment strategies for solid tumors in clinics. However, the hypoxic tumor microenvironment induced by uncontrolled cancer cell proliferation significantly reduces the therapeutic efficacy of these strategies. Here, we rationally constructed indocyanine green (ICG)-loaded ultrasmall gold nanoclusters (Au NCs-ICG) as theranostic nanozymes for modulating tumor hypoxia and augmenting cancer PDT and RT, respectively. The constructed Au NC-ICG nanozymes with an ultrasmall particle size (∼1 nm) exhibited favorable renal clearance performance, high substrate affinity (K m ≈ 2 mM) and good catalase-like activity (V max ≈ 4.55 × 10 -3 mM s -1 ). In 4T1 tumor-bearing mouse models, high tumor accumulation of Au NC-ICG nanozymes was clearly visualized by near-infrared fluorescence, photoacoustic and computed tomography imaging, showing the potential for the monitoring and guidance of PDT and RT. In addition, the Au NCs-ICG nanozymes effectively decomposed intratumoral H 2 O 2 into O 2 for overcoming hypoxia and subsequently enhancing PDT and RT, respectively. Moreover, the inherent X-ray absorption capacity of Au NCs-ICG greatly deposited radiation energy within the tumor region and further improved cancer RT. The integration of multimodal imaging, tumor hypoxia regulation, and effective therapy into ultrasmall Au NCs-ICG nanozymes shows great potential for cancer theranostic applications.

Objective: Recent in vivo results have shown prominent tissue sparing effect of radiotherapy with ultra-high dose rates (FLASH) compared to conventional dose rates (CONV). Oxygen depletion has been proposed as the underlying mechanism, but in vitro data to support this have been lacking. The aim of the current study was to compare FLASH to CONV irradiation under different oxygen concentrations in vitro ., Methods: Prostate cancer cells were irradiated at different oxygen concentrations (relative partial pressure ranging between 1.6 and 20%) with a 10 MeV electron beam at a dose rate of either 600 Gy/s (FLASH) or 14 Gy/min (CONV), using a modified clinical linear accelerator. We evaluated the surviving fraction of cells using clonogenic assays after irradiation with doses ranging from 0 to 25 Gy., Results: Under normoxic conditions, no differences between FLASH and CONV irradiation were found. For hypoxic cells (1.6%), the radiation response was similar up to a dose of about 5-10 Gy, above which increased survival was shown for FLASH compared to CONV irradiation. The increased survival was shown to be significant at 18 Gy, and the effect was shown to depend on oxygen concentration., Conclusion: The in vitro FLASH effect depends on oxygen concentration. Further studies to characterize and optimize the use of FLASH in order to widen the therapeutic window are indicated., Advances in Knowledge: This paper shows in vitro evidence for the role of oxygen concentration underlying the difference between FLASH and CONV irradiation.

Introduction: Radiation-induced DNA damage occurs from direct and indirect effects. The induction is influenced by the physical characteristics of the radionuclide, especially its linear energy transfer. Hypoxia reduces the effect of irradiation treatment in tumor cells and leads to poor patient outcomes. High linear energy transfer emitters can overcome this obstacle. Our aim is to demonstrate the influence of hypoxia on the interaction of different radiation qualities with isolated DNA., Methods: PuC19 Plasmid DNA was irradiated with 223 Ra, 188 Re, 99m Tc and 99m Tc-labeled pyrene with and without DMSO under hypoxia or normoxic conditions. DNA damages in form of single-(SSB) and double-strand breaks (DSB) were analyzed by gel electrophoresis., Results: Radiation doses up to 200 Gy of 223 Ra, 188 Re and 99m Tc led to maximal yields of 80% SSB and 30%, 28% and 32% DSB, respectively. Hypoxia had minor effects on damages from 223 Ra, but caused a small enhancement in DSB for 188 Re and 99m Tc. DMSO prevented DSB completely and reduced SSB from the "free" radionuclides to comparable levels. DNA-binding 99m Tc-labeled pyrene induced less SSB and DSB compared to [ 99m Tc]TcO 4 - . However, the incubation with DMSO could prevent the SSB and DSB induction only to a minor extent., Conclusions: Hypoxia does not limit DNA damage induced by 223 Ra, 188 Re, 99m Tc and 99m Tc-labeled pyrene. Dose-dependent radiation effects were comparable for alpha-emitters and both high- and low-energy electron emitters. The radioprotection by DMSO was not influenced by hypoxia. The results indicate the contribution of mainly indirect radiation effects for 99m Tc, 188 Re and 223 Ra. 99m Tc-labeled pyrene caused direct DNA damages and Auger-electrons from 99m Tc-labeled pyrene are more effective than high-energy electrons or alpha particles., Advances in Knowledge: Without the consideration of DNA repair mechanisms, oxygen has no direct influence in radiation-induced DNA damages by different radiation qualities., Implications for Patient Care: The short-time stimulation with oxygen during patient radiation could have minor influence compared to constant oxygen flooding to overcome hypoxic barriers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Radiation-resistant hypoxic tumor areas continue to present a major limitation for successful tumor treatment. To overcome this radiation resistance, an oxygen-independent treatment is proposed using UVC-emitting LuPO 4 :Pr 3+ nanoparticles (NPs) and X rays. The uptake of the NPs as well as their effect on cell proliferation was investigated on A549 lung cancer cells by using inverted time-lapse microscopy and transmission electron microscopy. Furthermore, cytotoxicity of the combined treatment of X rays and LuPO 4 :Pr 3+ NPs was assessed under normoxic and hypoxic conditions using the colony formation assay. Transmission electron microscopy (TEM) images showed no NP uptake after 3 h, whereas after 24 h incubation an uptake of NPs was documented. LuPO 4 :Pr 3+ NPs alone caused a concentration-independent cell growth delay within the first 60 h of incubation. The combined treatment with UVC-emitting NPs and X rays reduced the radiation resistance of hypoxic cells by a factor of two to the level of cells under normoxic condition. LuPO 4 :Pr 3+ NPs cause an early growth delay but no cytotoxicity for the tested concentration. The combination of these NPs with X rays increases cytotoxicity of normoxic and hypoxic cancer cells. Hypoxic cells become sensitized to normoxic cell levels.

We collected initial quantitative information on the effects of high-dose carbon ( 12 C) ions compared to photons on vascular damage in anaplastic rat prostate tumors, with the goal of elucidating differences in response to high-LET radiation, using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Syngeneic R3327-AT1 rat prostate tumors received a single dose of either 16 or 37 Gy 12 C ions or 37 or 85 Gy 6 MV photons (iso-absorbed and iso-effective doses, respectively). The animals underwent DCE-MRI prior to, and on days 3, 7, 14 and 21 postirradiation. The extended Tofts model was used for pharmacokinetic analysis. At day 21, tumors were dissected and histologically examined. The results of this work showed the following: 1. 12 C ions led to stronger vascular changes compared to photons, independent of dose; 2. Tumor growth was comparable for all radiation doses and modalities until day 21; 3. Nonirradiated, rapidly growing control tumors showed a decrease in all pharmacokinetic parameters (area under the curve, K trans , v e , v p ) over time; 4. 12 C-ion-irradiated tumors showed an earlier increase in area under the curve and K trans than photon-irradiated tumors; 5. 12 C-ion irradiation resulted in more homogeneous parameter maps and histology compared to photons; and 6. 12 C-ion irradiation led to an increased microvascular density and decreased proliferation activity in a largely dose-independent manner compared to photons. Postirradiation changes related to 12 C ions and photons were detected using DCE-MRI, and correlated with histological parameters in an anaplastic experimental prostate tumor. In summary, this pilot study demonstrated that exposure to 12 C ions increased the perfusion and/or permeability faster and led to larger changes in DCE-MRI parameters resulting in increased vessel density and presumably less hypoxia at the end of the observation period when compared to photons. Within this study no differences were found between curative and sub-curative doses in either modality.

Purpose: To evaluate the usefulness of combined treatment with both continuous administration of a hypoxic cytotoxin, tirapazamine (TPZ) and mild temperature hyperthermia (MTH) in boron neutron capture therapy (BNCT) in terms of local tumor response and lung metastatic potential, referring to the response of intratumor quiescent (Q) cells. Materials and methods: B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. The tumors received reactor thermal neutron beam irradiation following the administration of a 10 B-carrier ( L-para -boronophenylalanine- 10 B (BPA) or sodium mercaptoundecahydrododecaborate- 10 B (BSH)) after single intraperitoneal injection of an acute hypoxia-releasing agent (nicotinamide), MTH (40 °C for 60 min), and 24-h continuous subcutaneous infusion of TPZ or combined treatment with both TPZ and MTH. Immediately after irradiation, cells from some tumors were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (=P + Q) tumor cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumor-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Results: BPA-BNCT increased the sensitivity of the total tumor cell population more than BSH-BNCT. However, the sensitivity of Q cells treated with BPA was lower than that of BSH-treated Q cells. With or without a 10 B-carrier, combination with continuously administered TPZ with or without MTH enhanced the sensitivity of the both total and Q cells, especially Q cells. Even without irradiation, nicotinamide treatment decreased the number of lung metastases. With irradiation, BPA-BNCT, especially in combination with combined treatment with both TPZ and MTH as well as nicotinamide treatment, showed the potential to reduce the number more than BSH-BNCT. Conclusion: BSH-BNCT combined with TPZ with or without MTH improved local tumor control, while BPA-BNCT in combination with both TPZ and MTH as well as nicotinamide is thought to reduce the number of lung metastases. It was elucidated that control of the chronic hypoxia-rich Q cell population in the primary solid tumor has the potential to impact the control of local tumors as a whole and that control of the acute hypoxia-rich total tumor cell population in the primary solid tumor has the potential to impact the control of lung metastases.

Background and purpose: Poly(ADP-ribose)polymerase-1 (PARP1) and DNA-dependent protein kinase (DNA-PK) play key roles in the repair of radiation-induced DNA double strand breaks, but it is unclear which is the preferred therapeutic target in radiotherapy. Here we compare small molecule inhibitors of both as radiosensitizers of head and neck squamous cell carcinoma (HNSCC) cell lines. Methods: Two PARP1 inhibitors (olaparib, veliparib) and two DNA-PK inhibitors (KU57788, IC87361) were tested in 14 HNSCC cell lines and two non-tumorigenic lines (HEK-293 and WI-38/Va-13), with drug exposure for 6 or 24 h post-irradiation, using regrowth assays. For three lines (UT-SCC-54C, -74B, -76B), radiosensitization was also assessed by clonogenic assay under oxia and acute (6 h) anoxia, and for 54C cells under chronic hypoxia (0.2% O 2 for 48 h). Relationships between sensitizer enhancement ratios (SER) and gene expression, assessed by RNA sequencing, were evaluated. Results: The inhibitors were minimally cytotoxic in the absence of radiation, with 74B and 54C cells the most sensitive to both olaparib and KU57788. Median SER values for each inhibitor at 1.1 µM were 1.12 (range 1.02-1.24) for olaparib, 1.08 (1.04-1.13) for veliparib, 1.35 (1.10-1.64) for IC87361 and 1.77 (1.41-2.38) for KU57788. The higher SER values for the DNA-PK inhibitors were observed with all cell lines (except HEK-293) and all concentrations tested and were confirmed by clonogenic assay. Radiosensitization by the DNA-PK inhibitors correlated with expression of SLFN11 mRNA. Radiosensitization by IC87361 and olaparib was significantly enhanced under acute anoxia and chronic hypoxia. Conclusions: The DNA-PK inhibitors KU57788 and IC87361 are more effective radiosensitizers than the PARP-1 inhibitors olaparib and veliparib at non-cytotoxic concentrations in HNSCC cell cultures and their activity is enhanced by SLFN11 and hypoxia.

Thermo-radiosensitisation is a promising approach for treatment of radio-resistant tumours such as those containing hypoxic subregions. Response prediction and treatment planning should account for tumour response heterogeneity, e.g. due to microenvironmental factors, and quantification of the biological effects induced. 3D tumour spheroids provide a physiological in vitro model of tumour response and a systems oncology framework for simulating spheroid response to radiation and hyperthermia is presented. Using a cellular automaton model, 3D oxygen diffusion, delivery of radiation and/or hyperthermia were simulated for many ([Formula: see text]) individual cells forming a spheroid. The iterative oxygen diffusion model was compared to an analytical oxygenation model and simulations were calibrated and validated against experimental data for irradiated (0-10 Gy) and/or heated (0-240 CEM 43 ) HCT116 spheroids. Despite comparable clonogenic survival, spheroid growth differed significantly following radiation or hyperthermia. This dynamic response was described well by the simulation ([Formula: see text] > 0.85). Heat-induced cell death was implemented as a fast, proliferation-independent process, allowing reoxygenation and repopulation, whereas radiation was modelled as proliferation-dependent mitotic catastrophe. This framework stands out both through its experimental validation and its novel ability to predict spheroid response to multimodality treatment. It provides a good description of response where biological dose-weighting based on clonogenic survival alone was insufficient.

Hypoxia, as a characteristic feature of solid tumor, can significantly adversely affect the outcomes of cancer radiotherapy (RT), photodynamic therapy, or chemotherapy. In this study, a strategy is developed to overcome tumor hypoxia-induced radiotherapy tolerance. Specifically, a novel two-dimensional Pd@Au bimetallic core-shell nanostructure (TPAN) was employed for the sustainable and robust production of O 2 in long-term via the catalysis of endogenous H 2 O 2 . Notably, the catalytic activity of TPAN could be enhanced via surface plasmon resonance (SPR) effect triggered by NIR-II laser irradiation, to enhance the O 2 production and thereby relieve tumor hypoxia. Thus, TPAN could enhance radiotherapy outcomes by three aspects: 1) NIR-II laser triggered SPR enhanced the catalysis of TPAN to produce O 2 for relieving tumor hypoxia; 2) high-Z element effect arising from Au and Pd to capture X-ray energy within the tumor; and 3) TPAN affording X-ray, photoacoustic, and NIR-II laser derived photothermal imaging, for precisely guiding cancer therapy, so as to reduce the side effects from irradiation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, we investigated the change in tumor microenvironment caused by semi-ablative high-dose irradiation and its implication on tumor cell survival, reoxygenation of hypoxic cells and repopulation in FSaII tumors grown subcutaneously in the hind legs of C3H mice. Tumors were exposed to 10-30 Gy of X-ray radiation in a single exposure, and the vascularity and blood perfusion were assessed based on the levels of CD31 expression and Hoechst 33342 perfusion, respectively. The tumor hypoxia was assessed by staining for pimonidazole adduct formation and the expression of hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9). Tumor cell survival was determined using in vivo-in vitro excision assay method. The proportion of hypoxic cells in the tumor was determined from the surviving cell fraction in tumors exposed to a test dose under aerobic and hypoxic conditions. Radiation expsoure markedly reduced the functional vascularity and blood perfusion, and profoundly increased the expression of HIF-1α and CA9 pointing to an increase in tumor hypoxia. The overall clonogenic cell survival progressively decreased during 2-5 days postirradiation, most likely due to the radiation-induced vascular dysfunction. In turn, the proportion of surviving hypoxic cells decreased over several days postirradiation, presumably due to reoxygenation of hypoxic cells. The oxygen supplied through small fractions of blood vessels that survived the high-dose exposure, together with a reduction of oxygen consumption due to massive cell death, appeared to be the cause of the reoxygenation of hypoxic cells. The surviving tumor cells then subsequently repopulated. The findings from this study using a murine tumor model suggest that the efficacy of stereotactic body radiotherapy (SBRT) and stereotactic radiosurgery (SRS) may be significantly improved by allowing an inter-fraction time for reoxygenation while avoiding repopulation.

The tumor hypoxic microenvironment (THME) has a profound impact on tumor progression, and modulation of the THME has become an essential strategy to promote photodynamic therapy (PDT). Here, an oxygen self-supplied nanodelivery system that is based on nanometal-organic frameworks (nMOFs) with embedded AuNPs (Au@ZIF-8) on the nMOF surface as a catalase (CAT)-like nanozyme and encapsulating Ce6 inside as a photosensitizer was found to mitigate tumor hypoxia and reinforce PDT. As soon as Au@ZIF-8 reaches the tumor site, the AuNP nanozyme can catalyze excessive H2O2 to produce O2 to alleviate tumor hypoxia, promoting the production of 1O2 with strong toxicity toward tumor cells under irradiation. Our study demonstrates that nMOFs embellished with a nanozyme have great potential for overcoming the THME for cancer therapeutics, which provides a facile strategy for accurate bioimaging and cancer therapy in vivo.

Treatment failure through radioresistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signaling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we demonstrated that hypoxia enhanced EGFR expression and sustained cell survival in SiHa, CAL 27 and A549 cells at both low and high cell desnities, while in MCF-7, MDA-MB-231 and HeLa cells, EGFR and cell survival were regulated by hypoxic treatment in a cell-density dependent manner: upregulated at low cell density and downregulated at high cell density. In MCF-7 and HeLa xenografts in nude mice, EGFR expression varied inversely with the pimonidazole level that was used as an indicator of hypoxia, accordant with the effect of hypoxia at high cell density in vitro. Hypoxia induced more remarkable cell autophagy at high cell density than at low cell density. Autophagy inhibitor 3MA, rather than proteasome inhibitor MG132 inhibited hypoxia-mediated EGFR loss and shifted cell death to cell survival in HeLa cells. The MCF7 cells' sensitivity to ionizing radiation (IR) under hypoxia was also conditional on the cell densities when the hypoxia treatment was introduced, inversely associated with the expression levels of EGFR. Altogether, hypoxia can decrease EGFR expression in some cell lines by enhancing autophagy at high cell density, leading to cell death and hypersensitivity to radiotherapy. This study may help to understand how hypoxia influences EGFR expression and radiosensitivity., (Copyright © 2018. Published by Elsevier Ltd.)

Methenamine (hexamethylenetetramine, hexamine, urotropine) is a compound discovered in 1859, which is still currently being used as a urinary antiseptic. Methenamine is highly soluble in water and polar solvents, and its molecular constitution is similar to adamantane compounds with tetrahedral cage like structure. In acidic conditions, methenamine decomposes to formaldehyde and ammonia. Recently, methenamine has gained a renewal of interest due to antibiotic-resistant bacteria urinary tract infections; interestingly, bacteria cannot gain resistance to formaldehyde. In 1968, David and Burkitt reported remarkable regression of four Burkitt Lymphoma patients in eight subjects who were treated with septicemine (a solution containing 6.3 g of methenamine iodomethylate and 1 g of methenamine sodium benzoate in 100 cc distilled water). Unfortunately, these striking observations did not gain interest in the medical community; despite experimental models that showed that methenamine synergized with hyperthermia, radiation, and chemotherapy to block cancer growth. As the hypoxic core of tumours have an acidic pH, it would be plausible to expect that methenamine would selectively target dormant, non-proliferative, and treatment-resistant cancer clones in large tumours. Moreover, previous data suggests that methenamine can be safely used intravenously and for treatment of infections of the central nervous system. It may therefore be an effective adjuvant in treatment of systemic cancers and glioblastoma., (© 2019 John Wiley & Sons Australia, Ltd.)

Purpose: Tumor hypoxia, often found in nonsmall cell lung cancer (NSCLC), implies an increased resistance to radiotherapy. Pretreatment assessment of tumor oxygenation is, therefore, warranted in these patients, as functional imaging of hypoxia could be used as a basis for dose painting. This study aimed at investigating the feasibility of using a method for calculating the dose required in hypoxic subvolumes segmented on 18 F-HX4 positron emission tomography (PET) imaging of NSCLC., Methods: Positron emission tomography imaging data based on the hypoxia tracer 18 F-HX4 of 19 NSCLC patients were included in the study. Normalized tracer uptake was converted to oxygen partial pressure (pO 2 ) and hypoxic target volumes (HTVs) were segmented using a threshold of 10 mmHg. Uniform doses required to overcome the hypoxic resistance in the target volumes were calculated based on a previously proposed method taking into account the effect of interfraction reoxygenation, for fractionation schedules ranging from extremely hypofractionated stereotactic body radiotherapy (SBRT) to conventionally fractionated radiotherapy., Results: Gross target volumes ranged between 6.2 and 859.6 cm 3 , and the hypoxic fraction < 10 mmHg between 1.2% and 72.4%. The calculated doses for overcoming the resistance of cells in the HTVs were comparable to those currently prescribed in clinical practice as well as those previously tested in feasibility studies on dose escalation in NSCLC. Depending on the size of the HTV and the distribution of pO 2 , HTV doses were calculated as 43.6-48.4 Gy for a three-fraction schedule, 51.7-57.6 Gy for five fractions, and 59.5-66.4 Gy for eight fractions. For patients in whom the HTV pO 2 distribution was more favorable, a lower dose was required despite a bigger volume. Tumor control probability was lower for single-fraction schedules, while higher levels of tumor control probability were found for schedules employing several fractions., Conclusions: The method to account for heterogeneous and dynamic hypoxia in target volume segmentation and dose prescription based on 18 F-HX4-PET imaging appears feasible in NSCLC patients. The distribution of oxygen partial pressure within HTV could impact the required prescribed dose more than the size of the volume., (© 2019 American Association of Physicists in Medicine.)

The therapeutic potential of 177 Lu-iPSMA on hypoxic cancer cells has not been yet demonstrated. The aim of this work was to evaluate the radiation dose effect of 177 Lu-iPSMA on viability and DNA damage in U87MG human glioma cells subjected to hypoxia-mimetic conditions. U87MG cells treated with 177 Lu-iPSMA were incubated with CoCl 2 in order to induce hypoxia-mimetic conditions. The cytotoxic and genotoxic effect was evaluated with an in vitro viability test and a neutral comet assay. 177 Lu-iPSMA decreased the cell viability and induced DNA double strand breaks in U87MG human glioma cells under hypoxia-mimetic conditions. 177 Lu-iPSMA produced the maximum effect at 48 h, suggesting that this radiopharmaceutical could be used as a strategy for the treatment of human glioma hypoxic cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: It has been known for over 100 years that tumor hypoxia, a near-universal characteristic of solid tumors, decreases the curative effectiveness of radiation therapy. However, to date, there are no reports that demonstrate an improvement in radiation effectiveness in a mammalian tumor on the basis of tumor hypoxia localization and local hypoxia treatment., Methods and Materials: For radiation targeting of hypoxic subregions in mouse fibrosarcoma, we used oxygen images obtained using pulse electron paramagnetic resonance pO 2 imaging combined with 3D-printed radiation blocks. This achieved conformal radiation delivery to all hypoxic areas in FSa fibrosarcomas in mice., Results: We demonstrate that treatment delivering a radiation boost to hypoxic volumes has a significant (P = .04) doubling of tumor control relative to boosts to well-oxygenated volumes. Additional dose to well-oxygenated tumor regions minimally increases tumor control beyond the 15% control dose to the entire tumor. If we can identify portions of the tumor that are more resistant to radiation, it might be possible to reduce the dose to more sensitive tumor volumes without significant compromise in tumor control., Conclusions: This work demonstrates in a single, intact mammalian tumor type that tumor hypoxia is a local tumor phenomenon whose treatment can be enhanced by local radiation. Despite enormous clinical effort to overcome hypoxic radiation resistance, to our knowledge this is the first such demonstration, even in preclinical models, of targeting additional radiation to hypoxic tumor to improve the therapeutic ratio., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Strong oxygen dependence, poor tumor targeting, and limited treatment depth have been considered as the "Achilles' heels" facing the clinical usage of photodynamic therapy (PDT). Different from common approaches, here, we propose an innovative tactic by using photon-initiated dyad cationic superoxide radical (O 2 -• ) generator (ENBOS) featuring "0 + 1 > 1" amplification effect to simultaneously overcome these drawbacks. In particular, by taking advantage of the Förster resonance energy transfer theory, the energy donor successfully endows ENBOS with significantly enhanced NIR absorbance and photon utility, which in turn lead to ENBOS more easily activated and generating more O 2 -• in deep tissues, that thus dramatically intensifies the type I PDT against hypoxic deep tumors. Moreover, benefiting from the dyad cationic feature, ENBOS achieves superior "structure-inherent targeting" abilities with the signal-to-background ratio as high as 25.2 at 48 h post intravenous injection, offering opportunities for accurate imaging-guided tumor treatment. Meanwhile, the intratumoral accumulation and retention performance are also markedly improved (>120 h). On the basis of these unique merits, ENBOS selectively inhibits the deep-seated hypoxic tumor proliferation at a low light-dose irradiation. Therefore, this delicate design may open new horizons and cause a paradigm change for PDT in future cancer therapy.