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Enriched iron metabolic features such as high transferrin receptor (TfR) expression and high iron content are commonly observed in aggressive gliomas and can be associated with poor clinical responses. However, the underlying question of how iron contributes to tumor aggression remains elusive. Gliomas harboring isocitrate dehydrogenase (IDH) mutations account for a high percentage (>70 %) of recurrent tumors and cells with an acquired IDH mutation have been reported to have increased motility and invasion. This study aims to investigate how an acquired IDH mutation modulates iron metabolism and the implication(s) of iron on tumor cell growth. IDH mutant cells (U87 R132H ) grow significantly faster which is accompanied with increased TfR expression and iron uptake in vitro compared to wild-type U87 cells. This phenotype is retained in vivo. Biomechanically, U87 R132H cells are significantly less stiff and supplementation with ferrous ammonium sulfate (Fe 2+ ) augments membrane fluidity to drive U87 R132H cells into a super motile state. These findings provide insight into how an acquired IDH mutation may be able to modulate iron metabolism, allowing iron to serve as a biomechanical driver of tumor progression., Competing Interests: Declaration of competing interset The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gallium-based therapy has been considered a potentially effective cancer therapy for decades and has recently re-emerged as a novel therapeutic strategy for the management of glioblastoma tumors. Gallium targets the iron-dependent phenotype associated with aggressive tumors by mimicking iron in circulation and gaining intracellular access through transferrin-receptor-mediated endocytosis. Mechanistically, it is believed that gallium inhibits critical iron-dependent enzymes like ribonucleotide reductase and NADH dehydrogenase (electron transport chain complex I) by replacing iron and removing the ability to transfer electrons through the protein secondary structure. However, information regarding the effects of gallium on cellular iron metabolism is limited. As mitochondrial iron metabolism serves as a central hub of the iron metabolic network, the goal of this study was to investigate the effects of gallium on mitochondrial iron metabolism in glioblastoma cells. Here, it has been discovered that gallium nitrate can induce mitochondrial iron depletion, which is associated with the induction of DNA damage. Moreover, the generation of gallium-resistant cell lines reveals a highly unstable phenotype characterized by impaired colony formation associated with a significant decrease in mitochondrial iron content and loss of the mitochondrial iron uptake transporter, mitoferrin-1. Moreover, gallium-resistant cell lines are significantly more sensitive to radiation and have an impaired ability to repair any sublethal damage and to survive potentially lethal radiation damage when left for 24 h following radiation. These results support the hypothesis that gallium can disrupt mitochondrial iron metabolism and serve as a potential radiosensitizer.

Conventional cancer therapy strategies, although centered around killing tumor cells, often lead to severe side effects on surrounding normal tissues, thus compromising the chronic quality of life in cancer survivors. Hydrogen peroxide (H 2 O 2 ) is a secondary signaling molecule that has an array of functions in both tumor and normal cells, including the promotion of cell survival pathways and immune cell modulation in the tumor microenvironment. H 2 O 2 is a reactive oxygen species (ROS) crucial in cellular homeostasis and signaling (at concentrations maintained under nM levels), with increased steady-state levels in tumors relative to their normal tissue counterparts. Increased steady-state levels of H 2 O 2 in tumor cells, make them vulnerable to oxidative stress and ultimately, cell death. Recently, H 2 O 2 -producing therapies-namely, pharmacological ascorbate and superoxide dismutase mimetics-have emerged as compelling complementary treatment strategies in cancer. Both pharmacological ascorbate and superoxide dismutase mimetics can generate excess H 2 O 2 to overwhelm the impaired H 2 O 2 removal capacity of cancer cells. This review presents an overview of H 2 O 2 metabolism in the physiological and malignant states, in addition to discussing the anti-tumor and normal tissue-sparing mechanism(s) of, and clinical evidence for, two H 2 O 2 -based therapies, pharmacological ascorbate and superoxide dismutase mimetics.

The MR transverse relaxation rate, R2*, has been widely used to detect iron and myelin content in tissue. However, it is also sensitive to macroscopic B 0 inhomogeneities. One approach to correct for the B 0 effect is to fit gradient-echo signals with the three-parameter model, a sinc function-weighted monoexponential decay. However, such three-parameter models are subject to increased noise sensitivity. To address this issue, this study presents a two-stage fitting procedure based on the three-parameter model to mitigate the B 0 effect and reduce the noise sensitivity of R2* measurement in the mouse brain at 7T. MRI scans were performed on eight healthy mice. The gradient-echo signals were fitted with the two-stage fitting procedure to generate R2corr_t*. The signals were also fitted with the monoexponential and three-parameter models to generate R2nocorr* and R2corr*, respectively. Regions of interest (ROIs), including the corpus callosum, internal capsule, somatosensory cortex, caudo-putamen, thalamus, and lateral ventricle, were selected to evaluate the within-ROI mean and standard deviation (SD) of the R2* measurements. The results showed that the Akaike information criterion of the monoexponential model was significantly reduced by using the three-parameter model in the selected ROIs ( p = 0.0039-0.0078). However, the within-ROI SD of R2corr* using the three-parameter model was significantly higher than that of the R2nocorr* in the internal capsule, caudo-putamen, and thalamus regions ( p = 0.0039), a consequence partially due to the increased noise sensitivity of the three-parameter model. With the two-stage fitting procedure, the within-ROI SD of R2corr* was significantly reduced by 7.7-30.2% in all ROIs, except for the somatosensory cortex region with a fast in-plane variation of the B 0 gradient field ( p = 0.0039-0.0078). These results support the utilization of the two-stage fitting procedure to mitigate the B 0 effect and reduce noise sensitivity for R2* measurement in the mouse brain.

Soft tissue sarcomas (STSs) are mesenchymal malignant lesions that develop in soft tissues. Despite current treatments, including radiation therapy (RT) and surgery, STSs can be associated with poor patient outcomes and metastatic recurrences. Neoadjuvant radiation therapy (nRT), while effective, is often accompanied by severe postoperative wound healing complications due to damage to the surrounding normal tissues. Thus, there is a need to develop therapeutic approaches to reduce nRT toxicities. Avasopasem manganese (AVA) is a selective superoxide dismutase mimetic that protects against IR-induced oral mucositis and lung fibrosis. We tested the efficacy of AVA in enhancing RT in STSs and in promoting wound healing. Using colony formation assays and alkaline comet assays, we report that AVA selectively enhanced the STS (liposarcoma, fibrosarcoma, leiomyosarcoma, and MPNST) cellular response to radiation compared to normal dermal fibroblasts (NDFs). AVA is believed to selectively enhance radiation therapy by targeting differential hydrogen peroxide clearance in tumor cells compared to non-malignant cells. STS cells demonstrated increased catalase protein levels and activity compared to normal fibroblasts. Additionally, NDFs showed significantly higher levels of GPx1 activity compared to STSs. The depletion of glutathione using buthionine sulfoximine (BSO) sensitized the NDF cells to AVA, suggesting that GPx1 may, in part, facilitate the selective toxicity of AVA. Finally, AVA significantly accelerated wound closure in a murine model of wound healing post RT. Our data suggest that AVA may be a promising combination strategy for nRT therapy in STSs.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T 2 * MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T 2 * relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T 2 * mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

Background: Radiation therapy (RT) is an integral and commonly used therapeutic modality for primary lung cancer. However, radiation-induced lung injury (RILI) limits the irradiation dose used in the lung and is a significant source of morbidity. Disruptions in iron metabolism have been linked to radiation injury, but the underlying mechanisms remain unclear., Purpose: To utilize a targeted radiation delivery approach to induce RILI for the development of a model system to study the role of radiation-induced iron accumulation in RILI., Methods: This study utilizes a Small Animal Radiation Research Platform (SARRP) to target the right lung with a 20 Gy dose while minimizing the dose delivered to the left lung and adjacent heart. Long-term pulmonary function was performed using RespiRate-x64image analysis. Normal-appearing lung volumes were calculated using a cone beam CT (CBCT) image thresholding approach in 3D Slicer software. Quantification of iron accumulation was performed spectrophotometrically using a ferrozine-based assay as well as histologically using Prussian blue and via Western blotting for ferritin heavy chain expression., Results: Mild fibrosis was seen histologically in the irradiated lung using hematoxylin and eosin-stained fixed tissue at 9 months, as well as using a scoring system from CBCT images, the Szapiel scoring system, and the highest fibrotic area metric. In contrast, no changes in breathing rate were observed, and median survival was not achieved up to 36 weeks following irradiation, consistent with mild lung fibrosis when only one lung was targeted. Our study provided preliminary evidence on increased iron content and ferritin heavy chain expression in the irradiated lung, thus warranting further investigation., Conclusions: A targeted lung irradiation model may be a useful approach for studying the long-term pathological effects associated with iron accumulation and RILI following ionizing radiation.

Background: Pharmacological ascorbate (intravenous delivery reaching plasma concentrations ≈ 20 mM; P-AscH - ) has emerged as a promising therapeutic strategy for glioblastoma. Recently, a single-arm phase 2 clinical trial demonstrated a significant increase in overall survival when P-AscH - was combined with temozolomide and radiotherapy. As P-AscH - relies on iron-dependent mechanisms, this study aimed to assess the predictive potential of both molecular and imaging-based iron-related markers to enhance the personalization of P-AscH - therapy in glioblastoma participants., Methods: Participants (n = 55) with newly diagnosed glioblastoma were enrolled in a phase 2 clinical trial conducted at the University of Iowa (NCT02344355). Tumor samples obtained during surgical resection were processed and stained for transferrin receptor and ferritin heavy chain expression. A blinded pathologist performed pathological assessment. Quantitative susceptibility mapping (QSM) measures were obtained from pre-radiotherapy MRI scans following maximal safe surgical resection. Circulating blood iron panels were evaluated prior to therapy through the University of Iowa Diagnostic Laboratory., Results: Through univariate analysis, a significant inverse association was observed between tumor transferrin receptor expression and overall and progression-free survival. QSM measures exhibited a significant, positive association with progression-free survival. Subjects were actively followed until disease progression and then were followed through chart review or clinical visits for overall survival., Conclusions: This study analyzes iron-related biomarkers in the context of P-AscH - therapy for glioblastoma. Integrating molecular, systemic, and imaging-based markers offers a multifaceted approach to tailoring treatment strategies, thereby contributing to improved patient outcomes and advancing the field of glioblastoma therapy., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Purpose: Pharmacologic ascorbate (P-AscH-) is hypothesized to be an iron (Fe)-dependent tumor-specific adjuvant to chemoradiation in treating glioblastoma (GBM). This study determined the efficacy of combining P-AscH- with radiation and temozolomide in a phase II clinical trial while simultaneously investigating a mechanism-based, noninvasive biomarker in T2* mapping to predict GBM response to P-AscH- in humans., Patients and Methods: The single-arm phase II clinical trial (NCT02344355) enrolled 55 subjects, with analysis performed 12 months following the completion of treatment. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared across patient subgroups with log-rank tests. Forty-nine of 55 subjects were evaluated using T2*-based MRI to assess its utility as an Fe-dependent biomarker., Results: Median OS was estimated to be 19.6 months [90% confidence interval (CI), 15.7-26.5 months], a statistically significant increase compared with historic control patients (14.6 months). Subjects with initial T2* relaxation < 50 ms were associated with a significant increase in PFS compared with T2*-high subjects (11.2 months vs. 5.7 months, P < 0.05) and a trend toward increased OS (26.5 months vs. 17.5 months). These results were validated in preclinical in vitro and in vivo model systems., Conclusions: P-AscH- combined with temozolomide and radiotherapy has the potential to significantly enhance GBM survival. T2*-based MRI assessment of tumor iron content is a prognostic biomarker for GBM clinical outcomes. See related commentary by Nabavizadeh and Bagley, p. 255., (©2023 American Association for Cancer Research.)

Recently, the FDA-approved iron oxide nanoparticle, ferumoxytol, has been found to enhance the efficacy of pharmacological ascorbate (AscH - ) in treating glioblastoma, as AscH - reduces the Fe 3+ sites in the nanoparticle core. Given the iron oxidation state specificity of T2* relaxation mapping, this study aims to investigate the ability of T2* relaxation to monitor the reduction of ferumoxytol by AscH - with respect to its in vitro therapeutic enhancement. This study employed an in vitro glioblastoma MRI model system to investigate the chemical interaction of ferumoxytol with T 2 * mapping. Lipofectamine was utilized to facilitate ferumoxytol internalization and assess intracellular versus extracellular chemistry. In vitro T 2 * mapping successfully detected an AscH - -mediated reduction of ferumoxytol (25.6 ms versus 2.8 ms for FMX alone). The T 2 * relaxation technique identified the release of Fe 2+ from ferumoxytol by AscH - in glioblastoma cells. However, the high iron content of ferumoxytol limited T2* ability to differentiate between the external and internal reduction of ferumoxytol by AscH - (ΔT 2 * = +839% for external FMX and +1112% for internal FMX reduction). Notably, the internalization of ferumoxytol significantly enhances its ability to promote AscH - toxicity (dose enhancement ratio for extracellular FMX = 1.16 versus 1.54 for intracellular FMX). These data provide valuable insights into the MR-based nanotheranostic application of ferumoxytol and AscH - therapy for glioblastoma management. Future developmental efforts, such as FMX surface modifications, may be warranted to enhance this approach further.

Glioblastoma (GBM), a highly lethal and aggressive central nervous system malignancy, presents a critical need for targeted therapeutic approaches to improve patient outcomes in conjunction with standard-of-care (SOC) treatment. Molecular subtyping based on genetic profiles and metabolic characteristics has advanced our understanding of GBM to better predict its evolution, mechanisms, and treatment regimens. Pharmacological ascorbate (P-AscH - ) has emerged as a promising supplementary cancer therapy, leveraging its pro-oxidant properties to selectively kill malignant cells when combined with SOC. Given the clinical challenges posed by the heterogeneity and resistance of various GBM subtypes to conventional SOC, our study assessed the response of classical, mesenchymal, and proneural GBM to P-AscH - . P-AscH - (20 pmol/cell) combined with SOC (5 µM temozolomide and 4 Gy of radiation) enhanced clonogenic cell killing in classical and mesenchymal GBM subtypes, with limited effects in the proneural subtype. Similarly, following exposure to P-AscH - (20 pmol/cell), single-strand DNA damage significantly increased in classical and mesenchymal but not proneural GBM. Moreover, proneural GBM exhibited increased hydrogen peroxide removal rates, along with increased catalase and glutathione peroxidase activities compared to mesenchymal and classical GBM, demonstrating an altered H 2 O 2 metabolism that potentially drives differential P-AscH - toxicity. Taken together, these data suggest that P-AscH - may hold promise as an approach to improve SOC responsiveness in mesenchymal GBMs that are known for their resistance to SOC.

Background: Extremity soft-tissue sarcomas (STS) are commonly treated with neoadjuvant radiation therapy followed by surgical resection. However, the pathological near-complete response rate is low (9-25%). Noninvasive imaging assessment that predicts treatment response before and during treatment is desirable to optimize treatment regimens. This pilot study aimed to investigate the application of a quantitative MRI parameter, T 2 *, in assessing neoadjuvant radiation therapy combined with pharmacological ascorbate in extremity STS., Methods: This prospective cohort study included seven patients diagnosed with extremity STS and scheduled to receive neoadjuvant radiation therapy combined with pharmacological ascorbate. T 2 * maps were obtained from each patient before treatment (baseline MRI), two weeks after initiating treatment (on-treatment MRI), and before surgery (pre-surgery MRI). The T 2 * values within the tumor region were transformed into z-scores with respect to the normal- appearing tissue region. The voxel-wise z-scores within the tumor region were thresholded to generate masks representing significantly high (z-score>1.96) and low z-score (z-score<-1.96) voxels. The means of the total z-scores and within each of the significantly high and low z-score mask were computed. Their correlations with percent necrosis from pathological examination were evaluated using Spearman's rank correlation coefficient r. A correlation was considered as moderate or strong when r is higher than 0.6 and 0.8, respectively. A correlation was considered as fair or weak when r is below 0.6., Results: For the baseline and on-treatment MRIs, the means of the significantly high z-scores of the T 2 * measurements showed moderate correlations with percent necrosis (r = 0.68 and 0.6; p = 0.11 and 0.24). For the pre-surgery MRI, the means of the total and significantly high z-scores showed strong correlations with percent necrosis (r = 0.8 and 0.9; p = 0.13 and 0.08). Tumor volume and baseline MRI-based percent necrosis showed fair or weak correlations (r = 0.3-0.54; p = 0.24-0.68)., Conclusion: T 2 * measurements prior to treatment, two weeks after initiating treatment, and before surgery showed moderate to strong correlations with percent necrosis. These results support the potential for using T 2 * mapping to predict and assess response to neoadjuvant radiation therapy combined with pharmacological ascorbate in extremity STS. Level of Evidence: IV ., Competing Interests: Disclosures: The authors report no potential conflicts of interest related to this study., (Copyright © The Iowa Orthopaedic Journal 2023.)

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

A distinctive feature of cancer is the upregulation of sirtuin proteins. Sirtuins are class III NAD+-dependent deacetylases involved in cellular processes such as proliferation and protection against oxidative stress. SIRTs 1 and 2 are also overexpressed in several types of cancers including non-small cell lung cancer (NSCLC). Sirtinol, a sirtuin (SIRT) 1 and 2 specific inhibitor, is a recent anti-cancer agent that is cytotoxic against several types of cancers including NSCLC. Thus, sirtuins 1 and 2 represent valuable targets for cancer therapy. Recent studies show that sirtinol functions as a tridentate iron chelator by binding Fe3+ with 3:1 stoichiometry. However, the biological consequences of this function remain unexplored. Consistent with preliminary literature, we show that sirtinol can deplete intracellular labile iron pools in both A549 and H1299 non-small cell lung cancer cells acutely. Interestingly, a temporal adaptive response occurs in A549 cells as sirtinol enhances transferrin receptor stability and represses ferritin heavy chain translation through impaired aconitase activity and apparent IRP1 activation. This effect was not observed in H1299 cells. Holo-transferrin supplementation significantly enhanced colony formation in A549 cells while increasing sirtinol toxicity. This effect was not observed in H1299 cells. The results highlight the fundamental genetic differences that may exist between H1299 and A549 cells and offer a novel mechanism of how sirtinol kills NSCLC cells., 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 © 2023 Petronek, Bayanbold, Amegble, Tomanek-Chalkley, Allen, Spitz and Bailey.)

Ferumoxytol (FMX) is an FDA-approved magnetite (Fe 3 O 4 ) nanoparticle used to treat iron deficiency anemia that can also be used as an MR imaging agent in patients that can't receive gadolinium. Pharmacological ascorbate (P-AscH-; IV delivery; plasma levels ≈ 20 mM) has shown promise as an adjuvant to standard of care chemo-radiotherapy in glioblastoma (GBM). Since ascorbate toxicity mediated by H 2 O 2 is enhanced by Fe redox cycling, the current study determined if ascorbate catalyzed the release of ferrous iron (Fe 2+ ) from FMX for enhancing GBM responses to chemo-radiotherapy. Ascorbate interacted with Fe 3 O 4 in FMX to produce redox-active Fe 2+ while simultaneously generating increased H 2 O 2 fluxes, that selectively enhanced GBM cell killing (relative to normal human astrocytes) as opposed to a more catalytically active Fe complex (EDTA-Fe 3+ ) in an H 2 O 2 - dependent manner. In vivo, FMX was able to improve GBM xenograft tumor control when combined with pharmacological ascorbate and chemoradiation in U251 tumors that were unresponsive to pharmacological ascorbate therapy. These data support the hypothesis that FMX combined with P-AscH- represents a novel combined modality therapeutic approach to enhance cancer cell selective chemoradiosentization in the management of glioblastoma., Competing Interests: Declaration of competing interest This manuscript has not been published and is not under consideration for publication in other journals. We have no conflicts of interest to disclose., (Published by Elsevier B.V.)

Background: Ultrahigh dose-rate radiotherapy (FLASH-RT) affords improvements in the therapeutic index by minimizing normal tissue toxicities without compromising antitumor efficacy compared to conventional dose-rate radiotherapy (CONV-RT). To investigate the translational potential of FLASH-RT to a human pediatric medulloblastoma brain tumor, we used a radiosensitive juvenile mouse model to assess adverse long-term neurological outcomes., Methods: Cohorts of 3-week-old male and female C57Bl/6 mice exposed to hypofractionated (2 × 10 Gy, FLASH-RT or CONV-RT) whole brain irradiation and unirradiated controls underwent behavioral testing to ascertain cognitive status four months posttreatment. Animals were sacrificed 6 months post-irradiation and tissues were analyzed for neurological and cerebrovascular decrements., Results: The neurological impact of FLASH-RT was analyzed over a 6-month follow-up. FLASH-RT ameliorated neurocognitive decrements induced by CONV-RT and preserved synaptic plasticity and integrity at the electrophysiological (long-term potentiation), molecular (synaptophysin), and structural (Bassoon/Homer-1 bouton) levels in multiple brain regions. The benefits of FLASH-RT were also linked to reduced neuroinflammation (activated microglia) and the preservation of the cerebrovascular structure, by maintaining aquaporin-4 levels and minimizing microglia colocalized to vessels., Conclusions: Hypofractionated FLASH-RT affords significant and long-term normal tissue protection in the radiosensitive juvenile mouse brain when compared to CONV-RT. The capability of FLASH-RT to preserve critical cognitive outcomes and electrophysiological properties over 6-months is noteworthy and highlights its potential for resolving long-standing complications faced by pediatric brain tumor survivors. While care must be exercised before clinical translation is realized, present findings document the marked benefits of FLASH-RT that extend from synapse to cognition and the microvasculature., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Head and neck squamous cell carcinoma (HNSCC) patients treated with high-dose cisplatin concurrently with radiotherapy (hdCis-RT) commonly suffer kidney injury leading to acute and chronic kidney disease (AKD and CKD, respectively). We conducted a retrospective analysis of renal function and kidney injury-related plasma biomarkers in a subset of HNSCC subjects receiving hdCis-RT in a double-blinded, placebo-controlled clinical trial (NCT02508389) evaluating the superoxide dismutase mimetic, avasopasem manganese (AVA), an investigational new drug. We found that 90 mg AVA treatment prevented a significant reduction in estimated glomerular filtration rate (eGFR) three months as well as six and twelve months after treatment compared to 30 mg AVA and placebo. Moreover, AVA treatment may have allowed renal repair in the first 22 days following cisplatin treatment as evidenced by an increase in epithelial growth factor (EGF), known to aid in renal recovery. An upward trend was also observed in plasma iron homeostasis proteins including total iron (Fe-blood) and iron saturation (Fe-saturation) in the 90 mg AVA group versus placebo. These data support the hypothesis that treatment with 90 mg AVA mitigates cisplatin-induced CKD by inhibiting hdCis-induced renal changes and promoting renal recovery., Competing Interests: Declaration of competing interest Drs. Spitz and Allen acknowledge support for their laboratory efforts from a sponsored research agreement from Galera Therapeutics, Inc. Dr. Beardsley is an employee of and owns stock in, Galera Therapeutics, Inc. Dr. Holmlund owns stock in Galera Therapeutics, Inc. No potential conflicts of interest were disclosed by the other authors., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: Ataxia telangiectasia mutated kinase (ATM) inhibitors are potent radiosensitizers that regulate DNA damage responses and redox metabolism, but they have not been translated clinically because of the potential for excess normal tissue toxicity. Pharmacologic ascorbate (P-AscH - ; intravenous administration achieving mM plasma concentrations) selectively enhances H 2 O 2 -induced oxidative stress and radiosensitization in tumors while acting as an antioxidant and mitigating radiation damage in normal tissues including the bowel. We hypothesized that P-AscH - could enhance the therapeutic index of ATM inhibitor-based chemoradiation by simultaneously enhancing the intended effects of ATM inhibitors in tumors and mitigating off-target effects in adjacent normal tissues., Methods and Materials: Clonogenic survival was assessed in human (human colon tumor [HCT]116, SW480, HT29) and murine (CT26, MC38) colorectal tumor lines and normal cells (human umbilical vein endothelial cell, FHs74) after radiation ± DNA repair inhibitors ± P-AscH - . Tumor growth delay was assessed in mice with HCT116 or MC38 tumors after fractionated radiation (5 Gy × 3) ± the ATM inhibitor KU60019 ± P-AscH - . Intestinal injury, oxidative damage, and transforming growth factor β immunoreactivity were quantified using immunohistochemistry after whole abdominal radiation (10 Gy) ± KU60019 ± P-AscH - . Cell cycle distribution and ATM subcellular localization were assessed using flow cytometry and immunohistochemistry. The role of intracellular H 2 O 2 fluxes was assessed using a stably expressed doxycycline-inducible catalase transgene., Results: KU60019 with P-AscH - enhanced radiosensitization in colorectal cancer models in vitro and in vivo by H 2 O 2 -dependent oxidative damage to proteins and enhanced DNA damage, abrogation of the postradiation G2 cell cycle checkpoint, and inhibition of ATM nuclear localization. In contrast, concurrent P-AscH - markedly reduced intestinal toxicity and oxidative damage with KU60019., Conclusions: We provide evidence that redox modulating drugs, such as P-AscH - , may facilitate the clinical translation of ATM inhibitors by enhancing tumor radiosensitization while simultaneously protecting normal tissues., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Iron-sulfur (Fe-S) clusters are unique, redox-active co-factors ubiquitous throughout cellular metabolism. Fe-S cluster synthesis, trafficking, and coordination result from highly coordinated, evolutionarily conserved biosynthetic processes. The initial Fe-S cluster synthesis occurs within the mitochondria; however, the maturation of Fe-S clusters culminating in their ultimate insertion into appropriate cytosolic/nuclear proteins is coordinated by a late-acting cytosolic iron-sulfur assembly (CIA) complex in the cytosol. Several nuclear proteins involved in DNA replication and repair interact with the CIA complex and contain Fe-S clusters necessary for proper enzymatic activity. Moreover, it is currently hypothesized that the late-acting CIA complex regulates the maintenance of genome integrity and is an integral feature of DNA metabolism. This review describes the late-acting CIA complex and several [4Fe-4S] DNA metabolic enzymes associated with maintaining genome stability., 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 © 2023 Petronek and Allen.)

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs., 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 © 2022 Zaher, Stephens, Miller, Hartwig, Stolwijk, Petronek, Zacharias, Wadas, Monga, Cullen, Furqan, Houtman, Varga, Spitz and Allen.)

Polyoxometalate nanoparticles (POMs) are a class of compounds made up of multiple transition metals linked together using oxygen atoms. POMs commonly include group 6 transition metals, with two of the most common forms using molybdenum and tungsten. POMs are suggested to exhibit antimicrobial effects. In this study, we developed two POM preparations to study anti-cancer activity. We found that Mo-POM (NH 4 )Mo 7 O 24 ) and W-POM (H 3 PW 12 O 40 ) have anti-cancer effects on glioblastoma cells. Both POMs induced morphological changes marked by membrane swelling and the presence of multinucleated cells that may indicate apoptosis induction along with impaired cell division. We also observed significant increases in lipid oxidation events, suggesting that POMs are redox-active and can catalyze detrimental oxidation events in glioblastoma cells. Here, we present preliminary indications that molybdenum polyoxometalate nanoparticles may act like ferrous iron to catalyze the oxidation of phospholipids. These preliminary results suggest that Mo-POMs (NH 4 )Mo 7 O 24 ) and W-POMs (H 3 PW 12 O 40 ) may warrant further investigation into their utility as adjunct cancer therapies.

Background: Cancer cells often have altered iron metabolism relative to non-malignant cells with increased transferrin receptor and ferritin expression. Targeting iron regulatory proteins as part of a cancer therapy regimen is currently being investigated in various malignancies. Anti-cancer therapies that exploit the differences in iron metabolism between malignant and non-malignant cells (e.g. pharmacological ascorbate and iron chelation therapy) have shown promise in various cancers, including glioblastoma, lung, and pancreas cancers. Non-invasive techniques that probe tissue iron metabolism may provide valuable information for the personalization of iron-based cancer therapies. T 2 * mapping is a clinically available MRI technique that assesses tissue iron content in the heart and liver. We aimed to investigate the capacity of T 2 * mapping to detect iron stores in soft tissue sarcomas (STS)., Methods: In this study, we evaluated T 2 * relaxation times ex vivo in five STS samples from subjects enrolled on a phase Ib/IIa clinical trial combining pharmacological ascorbate with neoadjuvant radiation therapy. Iron protein expression levels (ferritin, transferrin receptor, iron response protein 2) were evaluated by Western blot analysis. Bioinformatic data relating clinical outcomes in STS patients and iron protein expression levels were evaluated using the KMplotter database., Results: There was a high level of inter-subject variability in the expression of iron protein and T 2 * relaxation times. We identified that T 2 * relaxation time is capable of accurately detecting ferritin-heavy chain expression (r = -0.96) in these samples. Bioinformatic data acquired from the KMplot database revealed that transferrin receptor and iron-responsive protein 2 may be negative prognostic markers while ferritin expression may be a positive prognostic marker in the management of STS., Conclusion: These data suggest that targeting iron regulatory proteins may provide a therapeutic approach to enhance STS management. Additionally, T 2 * mapping has the potential to be used a clinically accessible, non-invasive marker of STS iron regulatory protein expression and influence cancer therapy decisions that warrants further investigation. Level of Evidence: IV ., (Copyright © The Iowa Orthopaedic Journal 2022.)

Ferumoxytol (FMX) is an iron oxide nanoparticle that is FDA approved for the treatment of iron deficiency anemia. FMX contains an Fe3O4 core. Currently, the redox chemistry of Fe3O4 nanoparticles remains relatively unexplored. FMX has recently gained interest as an anti-cancer agent. Ionizing radiation (IR) is a treatment modality employed to treat several types of cancer. Utilizing electron paramagnetic resonance (EPR) spectroscopy, we found that the products produced from the radiolysis of water can oxidize the Fe3O4 core of FMX. Because of the limited diffusion of the HO2• and HO• produced, these highly oxidizing species have little direct effect on FMX oxidation. We have determined that H2O2 is the primary oxidant of FMX. In the presence of labile Fe2+, we found that reducing species generated from the radiolysis of H2O are able to reduce the Fe3+ sites of the Fe3O4 core. Importantly, we also have shown that IR stimulates the release of ferric iron from FMX. Because of its release of iron, FMX may serve as an adjuvant to enhance radiotherapy., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Gallium is a tri-valent p-block metal that closely mimics tri-valent iron. Gallium is internalized into cells via transferrin receptor-mediated endocytosis. Both Ga-67 and Ga-68 are radionuclides that can be radiolabeled to various bioactive compounds for clinical imaging procedures to visualize tumors and sites of inflammation. High-dose ascorbate (pharmacological ascorbate) is an emergent glioblastoma therapy that enhances cancer cell-killing through iron-metabolic perturbations. We hypothesized that pharmacological ascorbate treatments might alter Ga-67 uptake in glioblastoma cells. We evaluated the in vitro ability of pharmacological ascorbate to alter gallium uptake in patient-derived glioblastoma cells with variable genetic backgrounds by co-incubating cells with Ga-67 ± pharmacological ascorbate. Surprisingly, we observed increased basal gallium uptake in the glioblastoma cells compared to normal human astrocytes. Further, pharmacological ascorbate treatment stimulated gallium uptake in glioblastoma cells while not affecting uptake in normal human astrocytes. This effect appears to be related to transient increases in transferrin receptor expression. Finally, pharmacological ascorbate treatment appears to stimulate gallium uptake in an iron metabolism-dependent manner. Further mechanistic experiments are required to evaluate the translational utility of ascorbate to impact gallium tumor imaging., Competing Interests: Conflict of interest None.

T 2 * relaxation is an intrinsic magnetic resonance imaging (MRI) parameter that is sensitive to local magnetic field inhomogeneities created by the deposition of endogenous paramagnetic material (e.g. iron). Recent studies suggest that T 2 * mapping is sensitive to iron oxidation state. In this study, we evaluate the spin state-dependence of T 2 * relaxation using T 2 * mapping. We experimentally tested this physical principle using a series of phantom experiments showing that T 2 * relaxation times are directly proportional to the spin magnetic moment of different transition metals along with their associated magnetic susceptibility. We previously showed that T 2 * relaxation time can detect the oxidation of Fe 2+ . In this paper, we demonstrate that T 2 * relaxation times are significantly longer for the diamagnetic, d 10 metal Ga 3+ , compared to the paramagnetic, d 5 metal Fe 3+ . We also show in a cell culture model that cells supplemented with Ga 3+ (S = 0) have a significantly longer relaxation time compared to cells supplemented with Fe 3+ (S = 5/2). These data support the hypothesis that dipole-dipole interactions between protons and electrons are driven by the strength of the electron spin magnetic moment in the surrounding environment giving rise to T 2 * relaxation., (© 2021. The Author(s).)

Interest in the use of pharmacological ascorbate as a treatment for cancer has increased considerably since it was introduced by Cameron and Pauling in the 1970s. Recently, pharmacological ascorbate has been used in preclinical and early-phase clinical trials as a selective radiation sensitizer in cancer. The results of these studies are promising. This review summarizes data on pharmacological ascorbate (1) as a safe and efficacious adjuvant to cancer therapy; (2) as a selective radiosensitizer of cancer via a mechanism involving hydrogen peroxide; and (3) as a radioprotector in normal tissues. Additionally, we present new data demonstrating the ability of pharmacological ascorbate to enhance radiation-induced DNA damage in glioblastoma cells, facilitating cancer cell death. We propose that pharmacological ascorbate may be a general radiosensitizer in cancer therapy and simultaneously a radioprotector of normal tissue.

Cancer cells preferentially accumulate iron (Fe) relative to non-malignant cells; however, the underlying rationale remains elusive. Iron-sulfur (Fe-S) clusters are critical cofactors that aid in a wide variety of cellular functions (e.g., DNA metabolism and electron transport). In this article, we theorize that a differential need for Fe-S biogenesis in tumor versus non-malignant cells underlies the Fe-dependent cell growth demand of cancer cells to promote cell division and survival by promoting genomic stability via Fe-S containing DNA metabolic enzymes. In this review, we outline the complex Fe-S biogenesis process and its potential upregulation in cancer. We also discuss three therapeutic strategies to target Fe-S biogenesis: (i) redox manipulation, (ii) Fe chelation, and (iii) Fe mimicry.

There is a rapidly growing body of literature supporting the notion that differential oxidative metabolism in cancer versus normal cells represents a metabolic frailty that can be exploited to open a therapeutic window into cancer therapy. These cancer cell-specific metabolic frailties may be amenable to manipulation with non-toxic small molecule redox active compounds traditionally thought to be antioxidants. In this review we describe the potential mechanisms and clinical applicability in cancer therapy of four small molecule redox active agents: melatonin, vitamin E, selenium, and vitamin C. Each has shown the potential to have pro-oxidant effects in cancer cells while retaining antioxidant activity in normal cells. This dichotomy can be exploited to improve responses to radiation and chemotherapy by opening a therapeutic window based on a testable biochemical rationale amenable to confirmation with biomarker studies during clinical trials. Thus, the unique pro-oxidant/antioxidant properties of melatonin, vitamin E, selenium, and vitamin C have the potential to act as effective adjuvants to traditional cancer therapies, thereby improving cancer patient outcomes., (Copyright © 2021. Published by Elsevier B.V.)

MR-linac technology enhances the precision of therapeutic radiation by clarifying the tumor-normal tissue interface and provides the potential for adaptive treatment planning. Accurate delineation of tumors on diagnostic magnetic resonance imaging (MRI) frequently requires gadolinium-based contrast agents (GBCAs). Despite generally being considered safe, previous literature suggests that GBCAs are capable of contrast-induced acute kidney injury (AKI). It is unclear if the risk for AKI is enhanced when GBCAs are administered concurrently with ionizing radiotherapy. During irradiation, gadolinium may be liberated from its chelator which may induce AKI. The goal of this work was to determine if radiation combined with GBCAs increased the incidence of AKI. Using a preclinical MRI-guided irradiation system, where MRI acquisitions and radiation delivery are performed in rapid succession, tumor-bearing mice with normal kidney function were injected with GBCA and treated with 2, 8 or 18 Gy irradiation. Renal function was assessed on days three and seven postirradiation to assess for AKI. No clinically relevant changes in blood urea nitrogen and creatinine were observed in any combination of GBCA and radiation dose. From these data, we conclude that GBCA in combination with radiation does not increase the risk for AKI in mice. Additional investigation of multiple doses of GBCA administered concurrently with irradiation is warranted to evaluate the risk of chronic kidney injury., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Pharmacological ascorbate (P-AscH - ) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe 3+ ) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe 2+ ). Because paramagnetic Fe 3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH - -induced changes in redox-active Fe was assessed as a biomarker for therapy response. Gel phantoms containing either Fe 3+ or Fe 2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH - plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH - infusion, post-P-AscH - infusion, and post-radiation (3-4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe 3+ and Fe 2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool. Phantoms containing Fe 3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe 2+ phantoms. Compared to pre-P-AscH - , GBM T2* and QSM imaging were significantly changed post-P-AscH - infusion consistent with conversion of Fe 3+ to Fe 2+ . No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH - inducing greater changes in tumor labile iron pools compared to the normal tissue. CONCLUSIONS: T2* and QSM MR-imaging responses are consistent with P-AscH - reducing Fe 3+ to Fe 2+ , selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH - -induced changes in redox-active iron., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: The majority of colorectal cancers are resistant to cancer immune checkpoint inhibitors. Ionizing radiation (IR) and several radiosensitizers, including PARP inhibitors, can enhance responsiveness to immune checkpoint inhibitors by potentially complementary mechanisms of action. We assessed the ability of radiation and PARP inhibition to induce proimmunogenic changes in tumor cells and enhance their in vivo responsiveness to anti-PD-1 antibodies., Methods and Materials: We performed a candidate drug screen and used flow cytometry to assess effects of the PARP inhibitor veliparib on IR-mediated changes in MHC-1 antigen presentation and surface localization of immune-modulating proteins including PD-L1 and calreticulin in colorectal cancer tumor models. Reverse transcription polymerase chain reaction was used to assess the effects of veliparib and radiation on the expression of proinflammatory and immunosuppressive cytokines. The ability of concurrent PARP inhibition and subablative doses of radiation therapy to enhance in vivo responsiveness to anti-PD-1 antibodies was assessed using unilateral flank-tumor models with or without T-cell depletion., Results: Veliparib was a potent radiosensitizer in both cell lines. Radiation increased surface localization of MHC-1 and PD-L1 in a dose-dependent manner, and veliparib pretreatment significantly enhanced these effects with high (8 Gy) but not with lower radiation doses. Enhancement of MHC-1 and PD-L1 surface localization by IR and IR+ veliparib remained significant 1, 3, and 7 days after treatment. IR significantly increased delayed tumoral expression of proinflammatory cytokines interferon-Ƴ and CXCL10 but had no significant effect on the expression of IL-6 or TGF-β. Concurrent administration of veliparib and subablative radiation therapy (8 Gy × 2) significantly prolonged anti-PD-1-mediated in vivo tumor growth delay and survival in both tumor models. Moreover, these effects were more pronounced in the microsatellite instability-mutated MC38 tumor model. Enhancement of anti-PD-1 mediated tumor growth delay with veliparib and IR was attenuated by CD8+ T-cell depletion., Conclusions: We provide preclinical evidence for a novel therapeutic strategy to enhance responsiveness of colorectal tumors to immune checkpoint inhibitors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Historically, patients with localized soft tissue sarcomas (STS) of the extremities would undergo limb amputation. It was subsequently determined that the addition of radiation therapy (RT) delivered prior to (neoadjuvant) or after (adjuvant) a limb-sparing surgical resection yielded equivalent survival outcomes to amputation in appropriate patients. Generally, neoadjuvant radiation offers decreased volume and dose of high-intensity radiation to normal tissue and increased chance of achieving negative surgical margins-but also increases wound healing complications when compared to adjuvant radiotherapy. This review elaborates on the current neoadjuvant/adjuvant RT approaches, wound healing complications in STS, and the potential application of novel radioprotective agents to minimize radiation-induced normal tissue toxicity.

Patients diagnosed with metastatic sarcoma have limited options for achieving both local and distant tumor control. While SBRT can achieve local control, distant response rates remain low. There is limited evidence demonstrating the safety and efficacy for combining SBRT with concurrent PD-1 checkpoint blockade in metastatic sarcoma. In this prospective case-series, we examined five patients with metastatic sarcoma on pembrolizumab treated concurrently with SBRT from July 1, 2016-October 30, 2018. Acute and chronic toxicity were recorded using Common Terminology Criteria for Adverse Events (CTCAE, version 5.0). SBRT-treated tumor control was assessed using Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). With median follow-up of 14.9 months, three patients with undifferentiated pleomorphic sarcoma, one with intimal, and one with chondroblastic osteosarcoma received SBRT with concurrent pembrolizumab to 10 sites of metastatic disease. No grade 5 toxicities were observed. There was a single incidence of transient grade 4 lymphopenia which resolved without intervention. Grade 3 toxicities included anemia, thrombocytopenia, lymphopenia and colitis. One tumor demonstrated local progression after SBRT, and all others remained stable or with response. In conclusion, combining SBRT with PD-1 inhibition appeared to be safe in this patient population. Expected high rates of treated-tumor local control after SBRT were observed. Two of five patients demonstrated either enhanced local tumor regression, or possible abscopal effect., (©2020 by Radiation Research Society. All rights of reproduction in any form reserved.)

For decades the field of radiation oncology has sought to improve the therapeutic ratio through innovations in physics, chemistry, and biology. To date, technological advancements in image guided beam delivery techniques have provided clinicians with their best options for improving this critical tool in cancer care. Medical physics has focused on the preferential targeting of tumors while minimizing the collateral dose to the surrounding normal tissues, yielding only incremental progress. However, recent developments involving ultra-high dose rate irradiation termed FLASH radiotherapy (FLASH-RT), that were initiated nearly 50 years ago, have stimulated a renaissance in the field of radiotherapy, long awaiting a breakthrough modality able to enhance therapeutic responses and limit normal tissue injury. Compared to conventional dose rates used clinically (0.1-0.2 Gy/s), FLASH can implement dose rates of electrons or X-rays in excess of 100 Gy/s. The implications of this ultra-fast delivery of dose are significant and need to be re-evaluated to appreciate the fundamental aspects underlying this seemingly unique radiobiology. The capability of FLASH to significantly spare normal tissue complications in multiple animal models, when compared to conventional rates of dose-delivery, while maintaining persistent growth inhibition of select tumor models has generated considerable excitement, as well as skepticism. Based on fundamental principles of radiation physics, radio-chemistry, and tumor vs. normal cell redox metabolism, this article presents a series of testable, biologically relevant hypotheses, which may help rationalize the differential effects of FLASH irradiation observed between normal tissue and tumors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Iron (Fe) is an essential element that plays a fundamental role in a wide range of cellular functions, including cellular proliferation, DNA synthesis, as well as DNA damage and repair. Because of these connections, iron has been strongly implicated in cancer development. Cancer cells frequently have changes in the expression of iron regulatory proteins. For example, cancer cells frequently upregulate transferrin (increasing uptake of iron) and down regulate ferroportin (decreasing efflux of intracellular iron). These changes increase the steady-state level of intracellular redox active iron, known as the labile iron pool (LIP). The LIP typically contains approximately 2% intracellular iron, which primarily exists as ferrous iron (Fe 2+ ). The LIP can readily contribute to oxidative distress within the cell through Fe 2+ -dioxygen and Fenton chemistries, generating the highly reactive hydroxyl radical (HO • ). Due to the reactive nature of the LIP, it can contribute to increased DNA damage. Mitochondrial dysfunction in cancer cells results in increased steady-state levels of hydrogen peroxide and superoxide along with other downstream reactive oxygen species. The increased presence of H 2 O 2 and O 2 •- can increase the LIP, contributing to increased mitochondrial uptake of iron as well as genetic instability. Thus, iron metabolism and labile iron pools may play a central role connecting the genetic mutational theories of cancer to the metabolic theories of cancer.

Based on encouraging results from several early-phase clinical trials, there is renewed interest in the use of pharmacological ascorbate (i.e., intravenous administration resulting in >≈10 m M plasma ascorbate concentrations) in combination with standard-of-care cancer treatments including radiation and/or chemotherapy. Under normal, healthy physiological conditions, humans maintain plasma ascorbate concentrations in the range of 40-80 l M . However, in vivo antitumor activity requires supraphysiological plasma concentrations on the order of ≈20 m M . The stability of ascorbate in whole blood has been well studied. The goal of this work was to determine the appropriate handling methods of blood samples, after treatment with pharmacological ascorbate, which allow for the optimal measurement of ascorbate in plasma for dosing verification. Our findings indicate that ascorbate concentrations (m M ) are relatively stable in whole blood collected in sodium heparin tubes and stored on ice (or at 4°C) for up to 24 h. After 24 h, ascorbate levels in plasma are relatively stable at 4°C for up to 72 h. At -20°C, plasma concentrations are relatively stable for 2-3 weeks, while at -80°C, ascorbate concentrations in plasma are stable for at least one month. In contrast, patient samples showed better stability when stored as whole blood compared to plasma at 4°C but increasing hemolysis over time may significantly skew ascorbate measurements. Additionally, patient samples can be reliably stored as plasma at -20°C for up to three weeks in either a frost-containing or frost-free environment. This information can guide the collection, processing and storage of clinical samples after pharmacological ascorbate infusions amenable to multi-center clinical trials.