Opposing tumor-cell-intrinsic and -extrinsic roles of the IRF1 transcription factor in antitumor immunity.

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

NRF2 Mediates Cellular Resistance to Transformation, Radiation, and Inflammation in Mice.

Nuclear factor erythroid 2-related factor 2 (NRF2) is recognized as a master transcription factor that regulates expression of numerous detoxifying and antioxidant cytoprotective genes. In fact, models of NRF2 deficiency indicate roles not only in redox regulation, but also in metabolism, inflammatory/autoimmune disease, cancer, and radioresistancy. Since ionizing radiation (IR) generates reactive oxygen species (ROS), it is not surprising it activates NRF2 pathways. However, unexpectedly, activation is often delayed for many days after the initial ROS burst. Here, we demonstrate that, as assayed by γ-H2AX staining, rapid DNA double strand break (DSB) formation by IR in primary mouse Nrf2-/- MEFs was not affected by loss of NRF2, and neither was DSB repair to any great extent. In spite of this, basal and IR-induced transformation was greatly enhanced, suggesting that NRF2 protects against late IR-induced genomic instability, at least in murine MEFs. Another possible IR- and NRF2-related event that could be altered is inflammation and NRF2 deficiency increased IR-induced NF-κB pro-inflammatory responses mostly late after exposure. The proclivity of NRF2 to restrain inflammation is also reflected in the reprogramming of tumor antigen-specific lymphocyte responses in mice where Nrf2 k.o. switches Th2 responses to Th1 polarity. Delayed NRF2 responses to IR may be critical for the immune transition from prooxidant inflammation to antioxidant healing as well as in driving cellular radioresistance and survival. Targeting NRF2 to reprogram immunity could be of considerable therapeutic benefit in radiation and immunotherapy.

Classes of Drugs that Mitigate Radiation Syndromes.

We previously reported several vignettes on types and classes of drugs able to mitigate acute and, in at least one case, late radiation syndromes in mice. Most of these had emerged from high throughput screening (HTS) of bioactive and chemical drug libraries using ionizing radiation-induced lymphocytic apoptosis as a readout. Here we report the full analysis of the HTS screen of libraries with 85,000 small molecule chemicals that identified 220 "hits." Most of these hits could be allocated by maximal common substructure analysis to one of 11 clusters each containing at least three active compounds. Further screening validated 23 compounds as being most active; 15 of these were cherry-picked based on drug availability and tested for their ability to mitigate acute hematopoietic radiation syndrome (H-ARS) in mice. Of these, five bore a 4-nitrophenylsulfonamide motif while 4 had a quinoline scaffold. All but two of the 15 significantly (p < 0.05) mitigated H-ARS in mice. We had previously reported that the lead 4-(nitrophenylsulfonyl)-4-phenylpiperazine compound (NPSP512), was active in mitigating multiple acute and late radiation syndromes in mice of more than one sex and strain. Unfortunately, the formulation of this drug had to be changed for regulatory reasons and we report here on the synthesis and testing of active analogs of NPSP512 (QS1 and 52A1) that have increased solubility in water and in vivo bioavailability while retaining mitigator activity against H-ARS (p < 0.0001) and other radiation syndromes. The lead quinoline 057 was also active in multiple murine models of radiation damage. Taken together, HTS of a total of 150,000 bioactive or chemical substances, combined with maximal common substructure analysis has resulted in the discovery of diverse groups of compounds that can mitigate H-ARS and at least some of which can mitigate multiple radiation syndromes when given starting 24 h after exposure. We discuss what is known about how these agents might work, and the importance of formulation and bioavailability.

Weak Magnetic Fields Enhance the Efficacy of Radiation Therapy.

PURPOSE: The clinical efficacy of radiation therapy is mechanistically linked to ionization-induced free radicals that cause cell and tissue injury through direct and indirect mechanisms. Free radical reaction dynamics are influenced by many factors and can be manipulated by static weak magnetic fields (WMF) that perturb singlet-triplet state interconversion. Our study exploits this phenomenon to directly increase ionizing radiation (IR) dose absorption in tumors by combining WMF with radiation therapy as a new and effective method to improve treatment. METHODS AND MATERIALS: Coils were custom made to produce both homogeneous and gradient magnetic fields. The gradient coil enabled simultaneous in vitro assessment of free radical/reactive oxygen species reactivity across multiple field strengths from 6 to 66 G. First, increases in IR-induced free radical concentrations using oxidant-sensitive fluorescent dyes in a cell-free system were measured and verified. Next, human and murine cancer cell lines were evaluated in in vitro and in vivo models after exposure to clinically relevant doses of IR in combination with WMF. RESULTS: Cellular responses to IR and WMF were field strength and cell line dependent. WMF was able to enhance IR effects on reactive oxygen species formation, DNA double-strand break formation, cell death, and tumor growth. CONCLUSIONS: We demonstrate that the external presence of a magnetic field enhances radiation-induced cancer cell injury and death in vitro and in vivo. The effect extends beyond the timeframe when free radicals are induced in the presence of radiation into the window when endogenous free radicals are produced and therefore extends the applicability of this novel adjunct to cancer therapy in the context of radiation treatment.

Identification of miRNA signatures associated with radiation-induced late lung injury in mice.

Acute radiation exposure of the thorax can lead to late serious, and even life-threatening, pulmonary and cardiac damage. Sporadic in nature, late complications tend to be difficult to predict, which prompted this investigation into identifying non-invasive, tissue-specific biomarkers for the early detection of late radiation injury. Levels of circulating microRNA (miRNA) were measured in C3H and C57Bl/6 mice after whole thorax irradiation at doses yielding approximately 70% mortality in 120 or 180 days, respectively (LD70/120 or 180). Within the first two weeks after exposure, weight gain slowed compared to sham treated mice along with a temporary drop in white blood cell counts. 52% of C3H (33 of 64) and 72% of C57Bl/6 (46 of 64) irradiated mice died due to late radiation injury. Lung and heart damage, as assessed by computed tomography (CT) and histology at 150 (C3H mice) and 180 (C57Bl/6 mice) days, correlated well with the appearance of a local, miRNA signature in the lung and heart tissue of irradiated animals, consistent with inherent differences in the C3H and C57Bl/6 strains in their propensity for developing radiation-induced pneumonitis or fibrosis, respectively. Radiation-induced changes in the circulating miRNA profile were most prominent within the first 30 days after exposure and included miRNA known to regulate inflammation and fibrosis. Importantly, early changes in plasma miRNA expression predicted survival with reasonable accuracy (88-92%). The miRNA signature that predicted survival in C3H mice, including miR-34a-5p, -100-5p, and -150-5p, were associated with pro-inflammatory NF-κB-mediated signaling pathways, whereas the signature identified in C57Bl/6 mice (miR-34b-3p, -96-5p, and -802-5p) was associated with TGF-ß/SMAD signaling. This study supports the hypothesis that plasma miRNA profiles could be used to identify individuals at high risk of organ-specific late radiation damage, with applications for radiation oncology clinical practice or in the context of a radiological incident.

Association between Long-Term Second Malignancy Risk and Radiation: A Comprehensive Analysis of the Entire Surveillance, Epidemiology, and End Results Database (1973-2014).

PURPOSE: Second malignancies (SMs) after radiation therapy are rare but serious sequelae of treatment. This study investigates whether radiation therapy use is associated with changes in baseline SM risk. METHODS AND MATERIALS: We extracted all patients with cancer, with or without SM, in the Surveillance, Epidemiology, and End Results database from 1973 to 2014. Cumulative incidence of SM for patients stratified by radiation therapy status was calculated using a competing risk model, both for the entire cohort and for subgroups based on the primary tumor's anatomic location. RESULTS: We identified 2,872,063 patients with cancer, including 761,289 patients who received radiation therapy and 2,110,774 who did not. The SM rate at 20 years for patients receiving radiation therapy versus no radiation therapy was 21.4% versus 18.8%. The relative risk for SM associated with radiation therapy for the overall group was 1.138 at 20 years. The relative risks for SM associated with radiation therapy to malignancies arising from central nervous system and orbits, head and neck, thorax, abdomen, and pelvis at 20 years were 0.704, 1.011, 0.559, 0.646, and 1.106 for men and 0.792, 1.298, 1.265, 0.780, and 0.988 for women, respectively. CONCLUSIONS: The association between SM and radiation therapy varies with both sex and disease anatomic location, with the largest increase in SM seen in females irradiated to the head and neck region. Overall, the absolute change in SM rates associated with radiation therapy remains small, with differences in various clinical contexts.

The Aftermath of Surviving Acute Radiation Hematopoietic Syndrome and its Mitigation.

Intensive research is underway to find new agents that can successfully mitigate the acute effects of radiation exposure. This is primarily in response to potential counterthreats of radiological terrorism and nuclear accidents but there is some hope that they might also be of value for cancer patients treated with radiation therapy. Research into mitigation countermeasures typically employs classic animal models of acute radiation syndromes (ARS) that develop after whole-body irradiation (WBI). While agents are available that successfully mitigate ARS when given after radiation exposure, their success raises questions as to whether they simply delay lethality or unmask potentially lethal radiation pathologies that may appear later in time. Life shortening is a well-known consequence of WBI in humans and experimental animals, but it is not often examined in a mitigation setting and its causes, other than cancer, are not well-defined. This is in large part because delayed effects of acute radiation exposure (DEARE) do not follow the strict time-dose phenomena associated with ARS and present as a diverse range of symptoms and pathologies with low mortality rates that can be evaluated only with the use of large cohorts of subjects, as in this study. Here, we describe chronically increased mortality rates up to 660 days in large numbers of mice given LD70/30 doses of WBI. Systemic myeloid cell activation after WBI persists in some mice and is associated with late immunophenotypic changes and hematopoietic imbalance. Histopathological changes are largely of a chronic inflammatory nature and variable incidence, as are the clinical symptoms, including late diarrhea that correlates temporally with changes in the content of the microbiome. We also describe the acute and long-term consequences of mitigating hematopoietic ARS (H-ARS) lethality after LD70/30 doses of WBI in multiple cohorts of mice treated uniformly with radiation mitigators that have a common 4-nitro-phenylsulfonamide (NPS) pharmacophore. Effective NPS mitigators dramatically decrease ARS mortality. There is slightly increased subacute mortality, but the rate of late mortalities is slowed, allowing some mice to live a normal life span, which is not the case for WBI controls. The study has broad relevance to radiation late effects and their potential mitigation and epitomizes the complex interaction between radiation-damaged tissues and immune homeostasis.

Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation.

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.

4-(Nitrophenylsulfonyl)piperazines mitigate radiation damage to multiple tissues.

Our ability to use ionizing radiation as an energy source, as a therapeutic agent, and, unfortunately, as a weapon, has evolved tremendously over the past 120 years, yet our tool box to handle the consequences of accidental and unwanted radiation exposure remains very limited. We have identified a novel group of small molecule compounds with a 4-nitrophenylsulfonamide (NPS) backbone in common that dramatically decrease mortality from the hematopoietic acute radiation syndrome (hARS). The group emerged from an in vitro high throughput screen (HTS) for inhibitors of radiation-induced apoptosis. The lead compound also mitigates against death after local abdominal irradiation and after local thoracic irradiation (LTI) in models of subacute radiation pneumonitis and late radiation fibrosis. Mitigation of hARS is through activation of radiation-induced CD11b+Ly6G+Ly6C+ immature myeloid cells. This is consistent with the notion that myeloerythroid-restricted progenitors protect against WBI-induced lethality and extends the possible involvement of the myeloid lineage in radiation effects. The lead compound was active if given to mice before or after WBI and had some anti-tumor action, suggesting that these compounds may find broader applications to cancer radiation therapy.

Pattern of solid and hematopoietic second malignancy after local therapy for prostate cancer.

BACKGROUND AND PURPOSE: Second malignancies (SM) after external beam radiotherapy (EBRT) or brachytherapy (BT) for prostate cancer (PCa) are rare but serious sequelae. MATERIALS AND METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was used to identify men diagnosed with cT1-2N0M0 PCa between 1999 and 2005, who underwent EBRT, BT or radical prostatectomy (RP). Patients with time interval to second malignancy or follow-up shorter than five and two years were excluded for solid and hematopoietic SM analyses respectively. Risks for solid and hematopoietic SM were evaluated via the multivariate Fine and Gray proportional hazards model. RESULTS: EBRT and BT resulted in similar increases in solid and hematopoietic SM compared to RP. In subgroup analysis stratified by treatment modality, only the EBRT cohort demonstrated significantly decreased solid and hematopoietic SM in years 2002-2005 compared to years 1999-2001, with adjusted-hazard ratios of 0.752 (p=0.001) and 0.815 (p=0.018) respectively. CONCLUSIONS: EBRT and BT resulted in statistically equivalent increase in both solid and hematopoietic SM compared to RP. EBRT in more recent years resulted in significantly decreased solid and hematopoietic SM, coinciding with increased utilization of IMRT.

Investigation of DNA Damage Dose-Response Kinetics after Ionizing Radiation Schemes Similar to CT Protocols.

Although there has been extensive research done on the biological response to doses of ionizing radiation relevant to radiodiagnostic procedures, very few studies have examined radiation schemes similar to those frequently utilized in CT exams. Instead of a single exposure, CT exams are often made up of a series of scans separated on the order of minutes. DNA damage dose-response kinetics after radiation doses and schemes similar to CT protocols were established in both cultured (ESW-WT3) and whole blood lymphocytes and compared to higher dose exposures. Both the kinetics and extent of H2AX phosphorylation were found to be dose dependent. Damage induction and detection showed a clear dose response, albeit different, at all time points and differences in the DNA repair kinetics of ESW-WT3 and whole blood lymphocytes were characterized. Moreover, using a modified split-dose in vitro experiment, we show that phosphorylation of H2AX is significantly reduced after exposure to CT doses fractionated over a few minutes compared to the same total dose delivered as a single exposure. Because the split-dose exposures investigated here are more similar to those experienced during a CT examination, it is essential to understand why and how these differences occur. This work provides compelling evidence supporting differential biological responses not only between high and low doses, but also between single and multiple exposures to low doses of ionizing radiation.

Tumor control probability and the utility of 4D vs 3D dose calculations for stereotactic body radiotherapy for lung cancer.

Four-dimensional (4D) dose calculations for lung cancer radiotherapy have been technically feasible for a number of years but have not become standard clinical practice. The purpose of this study was to determine if clinically significant differences in tumor control probability (TCP) exist between 3D and 4D dose calculations so as to inform the decision whether 4D dose calculations should be used routinely for treatment planning. Radiotherapy plans for Stage I-II lung cancer were created for 8 patients. Clinically acceptable treatment plans were created with dose calculated on the end-exhale 4D computed tomography (CT) phase using a Monte Carlo algorithm. Dose was then projected onto the remaining 9 phases of 4D-CT using the Monte Carlo algorithm and accumulated onto the end-exhale phase using commercially available deformable registration software. The resulting dose-volume histograms (DVH) of the gross tumor volume (GTV), planning tumor volume (PTV), and PTVsetup were compared according to target coverage and dose. The PTVsetup was defined as a volume including the GTV and a margin for setup uncertainties but not for respiratory motion. TCPs resulting from these DVHs were estimated using a wide range of alphas, betas, and tumor cell densities. Differences of up to 5Gy were observed between 3D and 4D calculations for a PTV with highly irregular shape. When the TCP was calculated using the resulting DVHs for fractionation schedules typically used in stereotactic body radiation therapy (SBRT), the TCP differed at most by 5% between 4D and 3D cases, and in most cases, it was by less than 1%. We conclude that 4D dose calculations are not necessary for most cases treated with SBRT, but they might be valuable for irregularly shaped target volumes. If 4D calculations are used, 4D DVHs should be evaluated on volumes that include margin for setup uncertainty but not respiratory motion.

Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.

The high-affinity maltose switch MBP317-347 has low affinity for glucose: implications for targeting tumors with metabolically directed enzyme prodrug therapy.

Development of agents with high affinity and specificity for tumor-specific markers is an important goal of molecular-targeted therapy. Here, we propose a shift in paradigm using a strategy that relies on low affinity for fundamental metabolites found in different concentrations in cancerous and non-cancerous tissues: glucose and lactate. A molecular switch, MBP317-347, originally designed to be a high-affinity switch for maltose and maltose-like polysaccharides, was demonstrated to be a low-affinity switch for glucose, that is, able to be activated by high concentrations (tens of millimolar) of glucose. We propose that such a low-affinity glucose switch could be used as a proof of concept for a new prodrug therapy strategy denominated metabolically directed enzyme prodrug therapy (MDEPT) where glucose or, preferably, lactate serves as the activator. Accordingly, considering the typical differential concentrations of lactate found in tumors and in healthy tissues, a low-affinity lactate-binding switch analogous to the low-affinity glucose-binding switch MBP317-347 would be an order of magnitude more active in tumors than in normal tissues and therefore can work as a differential activator of anticancer drugs in tumors.

Factors affecting tumor (18) F-FDG uptake in longitudinal mouse PET studies.

BACKGROUND: Many biological factors of 2-[(18) F]fluoro-2-deoxy-d-glucose ((18) F-FDG) in blood can affect (18) F-FDG uptake in tumors. In this study, longitudinal (18) F-FDG positron emission tomography (PET) studies were performed on tumor-bearing mice to investigate the effect of blood glucose level and tumor size on (18) F-FDG uptake in tumors. METHODS: Six- to eight-week-old severe combined immunodeficiency mice were implanted with glioblastoma U87 (n = 8) or adenocarcinoma MDA-MB-231 (MDA) (n = 11) in the shoulder. When the tumor diameter was approximately 2.5 mm, a 60-min dynamic (18) F-FDG PET scan was performed weekly until the tumor diameter reached 10 mm. Regions of interests were defined in major organs and tumor. A plasma curve was derived based on a modeling method that utilizes the early heart time-activity curve and a late-time blood sample. The (18) F-FDG uptake constant K i was calculated using Patlak analysis on the tumors without an apparent necrotic center shown in the PET images. For each tumor type, the measured K i was corrected for partial volume (PV), and multivariate regression analysis was performed to examine the effects of blood glucose level ([Glc]) and tumor growth. Corrected Akaike's information criterion was used to determine the best model. RESULTS: The regression model that best fit the PV-corrected K i for U87 data was K i /RC = (1/[Glc]) × (0.27 ± 0.027) mL/min/mL (where [Glc] is in mmol/L), and for MDA, it was K i /RC = (0.04 ± 0.005) mL/min/mL, where K i /RC denotes the PV-corrected K i using an individual recovery coefficient (RC). The results indicated that (18) F-FDG K i /RC for U87 was inversely related to [Glc], while [Glc] had no effect on (18) F-FDG K i /RC of MDA. After the effects of PV and [Glc] were accounted for, the data did not support any increase of (18) F-FDG K i as the tumor (of either type) grew larger in size. CONCLUSIONS: The effect of [Glc] on the tumor (18) F-FDG K i was tumor-dependent. PV- and [Glc]-corrected (18) F-FDG K i did not show significant increase as the tumor of either type grew in size.

Re-evaluation of cellular radiosensitization by 5-fluorouracil: high-dose, pulsed administration is effective and preferable to conventional low-dose, chronic administration.

PURPOSE: It is widely believed that the anticancer drug 5- fluorouracil (5-FU) must be administered chronically and in low doses to maximize radiosensitization during chemoradiotherapy. The rationale is based upon cell experiments that assumed identical mechanisms of 5-FU action between low-dose chronic (LDC) and high-dose pulsed (HDP) exposures. Here we challenge the paradigm and demonstrate the effectiveness of HDP 5-FU as a radiosensitizer and the wide range of dose/time schedules that can be used to synergize with radiation as compared to the relatively restrictive protocols prescribed for current LDC administrations. MATERIALS AND METHODS: Clonogenic survival of human glioblastoma and colon cancer cell lines, U87MG-VIII and HCT-116, respectively, was used to assess temporal and dose effects of 5-FU on radiosensitivity and in split-dose experiments to characterize changes in sublethal damage repair. RESULTS: We show that HDP 5-FU administration does indeed radiosensitize both the highly radioresistant U87MG-VIII and HCT-116. Additionally, we show that this radiosensitization lasts for at least 24 h if cells are pre-irradiated with 2 Gy immediately after HDP 5-FU exposure as a result of a decrease in sublethal damage repair capacity for subsequent irradiations, suggesting the ideal combination of 5-FU bolus injection with fractionation radiotherapy schemes. CONCLUSIONS: 5-FU bolus administration protocols combined with radiation would not only help improve treatment outcomes and reduce development of 5-FU resistance, but it would greatly benefit patients by shortening clinical stays and lowering overall therapeutic costs.

Cellular autofluorescence following ionizing radiation.

Cells often autofluoresce in response to UV radiation excitation and this can reflect critical aspects of cellular metabolism. Here we report that many different human and murine cell types respond to ionizing radiation with a striking rise in autofluorescence that is dependent on dose and time. There was a highly reproducible fluorescent shift at various wavelengths, which was mirrored by an equally reproducible rise in the vital intracellular metabolic co-factors FAD and NADH. It appears that mitochondria, metabolism and Ca(2+) homeostasis are important for this to occur as cells without mitochondria or cells unable to alter calcium levels did not behave in this way. We believe these radiation-induced changes are of biological importance and that autofluorescence may even provide us with a tool to monitor radiation responses in the clinic.

Maximizing tumor immunity with fractionated radiation.

PURPOSE: Technologic advances have led to increased clinical use of higher-sized fractions of radiation dose and higher total doses. How these modify the pathways involved in tumor cell death, normal tissue response, and signaling to the immune system has been inadequately explored. Here we ask how radiation dose and fraction size affect antitumor immunity, the suppression thereof, and how this might relate to tumor control. METHODS AND MATERIALS: Mice bearing B16-OVA murine melanoma were treated with up to 15 Gy radiation given in various-size fractions, and tumor growth followed. The tumor-specific immune response in the spleen was assessed by interferon-γ enzyme-linked immunospot (ELISPOT) assay with ovalbumin (OVA) as the surrogate tumor antigen and the contribution of regulatory T cells (Tregs) determined by the proportion of CD4(+)CD25(hi)Foxp3(+) T cells. RESULTS: After single doses, tumor control increased with the size of radiation dose, as did the number of tumor-reactive T cells. This was offset at the highest dose by an increase in Treg representation. Fractionated treatment with medium-size radiation doses of 7.5 Gy/fraction gave the best tumor control and tumor immunity while maintaining low Treg numbers. CONCLUSIONS: Radiation can be an immune adjuvant, but the response varies with the size of dose per fraction. The ultimate challenge is to optimally integrate cancer immunotherapy into radiation therapy.

Radiation enhances regulatory T cell representation.

PURPOSE: Immunotherapy could be a useful adjunct to standard cytotoxic therapies such as radiation in patients with micrometastatic disease, although successful integration of immunotherapy into treatment protocols will require further understanding of how standard therapies affect the generation of antitumor immune responses. This study was undertaken to evaluate the impact of radiation therapy (RT) on immunosuppressive T regulatory (Treg) cells. METHODS AND MATERIALS: Treg cells were identified as a CD4(+)CD25(hi)Foxp3(+) lymphocyte subset, and their fate was followed in a murine TRAMP C1 model of prostate cancer in mice with and without RT. RESULTS: CD4(+)CD25(hi)Foxp3(+) Treg cells increased in immune organs after local leg or whole-body radiation. A large part, but not all, of this increase after leg-only irradiation could be ascribed to radiation scatter and Treg cells being intrinsically more radiation resistant than other lymphocyte subpopulations, resulting in their selection. Their functional activity on a per-cell basis was not affected by radiation exposure. Similar findings were made with mice receiving local RT to murine prostate tumors growing in the leg. The importance of the Treg cell population in the response to RT was shown by systemic elimination of Treg cells, which greatly enhanced radiation-induced tumor regression. CONCLUSIONS: We conclude that Treg cells are more resistant to radiation than other lymphocytes, resulting in their preferential increase. Treg cells may form an important homeostatic mechanism for tissues injured by radiation, and in a tumor context, they may assist in immune evasion during therapy. Targeting this population may allow enhancement of radiotherapeutic benefit through immune modulation.

High-throughput screening identifies two classes of antibiotics as radioprotectors: tetracyclines and fluoroquinolones.

PURPOSE: Discovery of agents that protect or mitigate normal tissue from radiation injury during radiotherapy, accidents, or terrorist attacks is of importance. Specifically, bone marrow insufficiency, with possible infection due to immunosuppression, can occur after total body irradiation (TBI) or regional irradiation and is a major component of the acute radiation syndrome. The purpose of this study was to identify novel radioprotectors and mitigators of the hematopoietic system. EXPERIMENTAL DESIGN: High-throughput screening of small-molecule libraries was done using viability of a murine lymphocyte line as a readout with further validation in human lymphoblastoid cells. The selected compounds were then tested for their ability to counter TBI lethality in mice. RESULTS: All of two major classes of antibiotics, tetracyclines and fluoroquinolones, which share a common planar ring moiety, were radioprotective. Furthermore, tetracycline protected murine hematopoietic stem/progenitor cell populations from radiation damage and allowed 87.5% of mice to survive when given before and 35% when given 24 h after lethal TBI. Interestingly, tetracycline did not alter the radiosensitivity of Lewis lung cancer cells. Tetracycline and ciprofloxacine also protected human lymphoblastoid cells, reducing radiation-induced DNA double-strand breaks by 33% and 21%, respectively. The effects of these agents on radiation lethality are not due to the classic mechanism of free radical scavenging but potentially through activation of the Tip60 histone acetyltransferase and altered chromatin structure. CONCLUSIONS: Tetracyclines and fluoroquinolones can be robust radioprotectors and mitigators of the hematopoietic system with potential utility in anticancer radiotherapy and radiation emergencies.