Radiation-induced Abscopal Effect Research Articles

Systemic effects of synchrotron radiation

A change in an organ or tissue distant from the irradiated region was termed the radiation-induced abscopal effect (RIAE). It is not known how radiation settings affect non-targeted normal tissues and therefore the risk of radiation-related adverse abscopal effects. In a recent study, we examined abscopal effects of microbeam radiotherapy (MRT) and broad beam (BB) configurations, in mice that were locally exposed to a very short pulse of a high dose-rate synchrotron beam utilizing the Imaging and Medical Beamline (IMBL) at the Australian Synchrotron. Here we summarise this study. Oxidative DNA damage was elevated in a wide variety of unirradiated normal tissues. Out-of-field duodenum showed a trend for elevated apoptotic cell death under most irradiation conditions, however, double-strand breaks (DSBs) elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in concentrations of several plasma cytokines and in frequencies of macrophages, neutrophils and T-lymphocytes in duodenum. Overall, systemic radiation responses were independent of dose, time post-irradiation, and radiation modality. These findings have implications for the planning of therapeutic and diagnostic radiation treatment to reduce the risk of radiation-related adverse systemic effects.

A Functional Immune System Is Required for the Systemic Genotoxic Effects of Localized Irradiation

Nontargeted effects of ionizing radiation, by which unirradiated cells and tissues are also damaged, are a relatively new paradigm in radiobiology. We recently reported radiation-induced abscopal effects (RIAEs) in normal tissues; namely, DNA damage, apoptosis, and activation of the local and systemic immune responses in C57BL6/J mice after irradiation of a small region of the body. High-dose-rate, synchrotron-generated broad beam or multiplanar x-ray microbeam radiation therapy was used with various field sizes and doses. This study explores components of the immune system involved in the generation of these abscopal effects. The following mice with various immune deficiencies were irradiated with the microbeam radiation therapy beam: (1) SCID/IL2γR-/- (NOD SCID gamma, NSG) mice, (2) wild-type C57BL6/J mice treated with an antibody-blocking macrophage colony-stimulating factor 1 receptor, which depletes and alters the function of macrophages, and (3) chemokine ligand 2/monocyte chemotactic protein 1 null mice. Complex DNA damage (ie, DNA double-strand breaks), oxidatively induced clustered DNA lesions, and apoptotic cells in tissues distant from the irradiation site were measured as RIAE endpoints and compared with those in wild-type C57BL6/J mice. Wild-type mice accumulated double-strand breaks, oxidatively induced clustered DNA lesions, and apoptosis, enforcing our RIAE model. However, these effects were completely or partially abrogated in mice with immune disruption, highlighting the pivotal role of the immune system in propagation of systemic genotoxic effects after localized irradiation. These results underline the importance of not only delineating the best strategies for tumor control but also mitigating systemic radiation toxicity.

Abstract 4573: Localized synchrotron radiation in mice induces persistent systemic genotoxic events mediated by the functional immune system

Abstract The discovery of the radiation-induced bystander effect has expanded knowledge of radiobiological mechanisms of ionizing radiation. A counterpart in vivo phenomenon is the radiation-induced abscopal effect (RIAE). It is not known how radiation settings affect non-targeted normal tissues and therefore the risk of adverse RIAE. At the Australian Synchrotron we examined systemic effects of microbeam radiotherapy (MRT) and broad beam (BB) configurations, in mice that were locally exposed to a very short pulse of a high dose-rate synchrotron beam (49 Gy/sec). We determined how radiation volume and dose impact the RIAE. C57BL/6 mice were irradiated with 10 or 40 Gy incident dose of MRT or BB in an 8x8, 8x1, or 2x2-mm area of the right hind leg. Blood samples, irradiated skin and a variety of normal unirradiated tissues were collected for DNA damage analysis of double-strand breaks (DSBs) quantified as gamma-H2AX foci and oxidatitive clustered DNA lesions (OCDL). OCDLs elevated in a wide variety of unirradiated normal tissues. In out-of-field duodenum, a trend for elevated apoptotic cells was observed, however DSBs elevated only after exposure to lower doses. These genotoxic events were accompanied by changes in concentrations of MDC, CCL2/MCP1, Eotaxin, IL-10, TIMP-1, VEGF, TGFβ-1 and TGFβ-2 plasma cytokines and by changes in frequencies of macrophages, neutrophils and T-lymphocytes in duodenum. Overall, systemic radiation responses were dose-independent (1). The MRT irradiations was repeated in immune-deficient mice: NSG, CCL2 knock-outs, and in C57BL/6 mice treated with anti-CSF1R antibody which effectively depletes macrophages. Strikingly, these effects and the abscopal innate and adaptive immune effector responses were completely or partially abrogated in the mice with various immune deficiencies, highlighting the role of the functional immune system in propagation of systemic genotoxic effects of radiation. These findings have implications for the planning of therapeutic and diagnostic radiation to reduce the risk of radiation-related adverse systemic effects. 1. Ventura et al, Cancer Research, e-pub (2017). Citation Format: Jessica Ventura, Pavel Lobachevsky, Jason Palazzolo, Helen Forrester, Nicole Haynes, Alesia Ivashkevich, Andrew Stevenson, Christopher Hall, Vassilis Gorgoulis, John Hamilton, Alexandros Georgakilas, Calr Sprung, Olga A. Martin. Localized synchrotron radiation in mice induces persistent systemic genotoxic events mediated by the functional immune system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4573.

Radiation and hypoxia-induced non-targeted effects in normoxic and hypoxic conditions in human lung cancer cells

Purpose: Many cell lines with anaerobic metabolism do not show cytotoxic abscopal effect (AE) following irradiation. Further, there is no existing data on the radiation- and hypoxia (H)-induced AE. The purpose of this study was to investigate and compare the status of radiation-induced abscopal effect (RIAE) in normoxic and hypoxic conditions.Methods: Lung cancer cells (A549, H460) were exposed either to hypoxia or normoxia and then irradiated (2 or 10 Gy). After 24 h, unirradiated hypoxic (H-CM) or normoxic (N-CM) conditioned media (CM) and irradiated hypoxic (H-RCM) or normoxic (N-RCM) CM was collected. Hypoxia-resistant clones (HR: A549/HR, H460/HR) were generated by continuous exposure of the cells to hypoxia. Unirradiated parental cells or HR were exposed to H-CM, N-CM, H-RCM or N-RCM. In some groups, 24 h after exposure to CM, cells were directly irradiated with 2 Gy. Cell growth was monitored using real-time cell electronic sensing system. Further, levels of hypoxia and HIF1α regulated angiogenesis related growth factors, basic fibroblast growth factor (bFGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt-1) and vascular endothelial growth factor (VEGF) were assessed in CM.Results: In the radio-resistant A549 cells, H-RCM was much more effective in inducing growth delay compared to N-RCM. In the radio-sensitive H460 cells, both N-RCM and H-RCM induced growth delay. Interestingly, effects of N-RCM were completely reversed in HR cells. Exposure of cells to direct irradiation (2 Gy) 24 h after incubation with CM resulted in 50–60% reduction in cell proliferation in A549/HR cells and a very significant induction of death (>95%) in H460/HR cells. Direct irradiation of parental or HR clones of A549 and H460 cells exposed to H-CM 24 h with 2 Gy induced significant reduction in cell proliferation (from 40% to >95%) in all the cells. Further, levels of sFlt-1 correlated with growth delay in all the cells.Conclusions: These results for the first time demonstrate that irradiation of hypoxic cells and exposing the cells to acute hypoxia lead to significant AE.

Radiation induced bystander effects in the spleen of cranially-irradiated rats.

To investigate the radiation-induced abscopal effect in terms of oxidative stress, apoptosis and DNA damage in the spleen cells following cranial X-rays irradiation of rats. Rats were cranially irradiated using 2 Gy X-rays. Another group was whole-body irradiated with 2 Gy X-rays and a third group was exposed to scattered radiation (measured to be 3 mGy). 24 hours following irradiation, sections from the spleen of the rats were dissected as well as plasma samples. The samples were examined for the desired endpoints. The cranially irradiated animals showed a significant increase in the levels of glutathione, superoxide dismutase and catalase with no significant change in the lipid peroxidation product in the spleen cells with a significant increase in the C-reactive protein level the plasma. Apoptotic cell death in the spleen cells was demonstrated as indicated by the decrease of Bcl-2; the increase of p53, Bax, caspase-3 and caspase-8 and induction of DNA damage in the spleen in both of the cranially irradiated rats and whole body exposed rats. The exposure to 3 mGy scattered radiation increased the plasma level of C-RP and also induced apoptosis in the spleen cells. Cranial irradiation-induced abscopal effect in distant spleen cells. Very low doses of radiation can induce apoptosis in the spleen cells. Advances in knowledge: This paper provides an evidence on the incidence of radiation abscopal effect. Also, the results shed light of the effect very low doses of radiation as low as 3 mGy.

EP-2037: Radiation-induced abscopal effect in normoxic and hypoxic conditions in lung adenocarcinoma

EP-2037: Radiation-induced abscopal effect in normoxic and hypoxic conditions in lung adenocarcinoma

Abstract A18: A systems biology approach to predict immunotherapy augmented abscopal effects

Abstract Introduction and Objectives: Radiation-induced abscopal effects are characterized by regression of tumor nodules outside the local radiation treatment field. Abscopal effects have been reported in metastatic lymphoma, melanoma, lung and breast cancer as well as renal cell carcinoma amongst others. Radiotherapy causes a perturbation to the local immune profile that is propagated throughout the lymphatic system, shifting systemic immune surveillance towards tumor eradication in distant, non-irradiated sites as well as circulating micrometastases. We hypothesize that abscopal effects could be triggered by targeting combined radiation and immunotherapy to immunogenic hubs in the patient-specific interconnected metastatic-lymphatic network. Materials and Methods: We develop a mathematical model of the systemic response to local radio-immunotherapy. Patient-specific positron emission tomography (PET) scans provide the size and location of each tumor nodule. Metastatic geography is mapped onto the lymphatic network and connections between metastatic sites and lymphatic nodes are calculated. We quantify immune signal flow through the lymphatic network and amplifications through metastatic sites. A local model describes the temporal coevolution of tumor size with effector and regulatory T cells. The local and global models are coupled to quantify systemic responses to local treatment of each tumor site. Results: The ‘virtual patient’ framework successfully simulates the abscopal effect reported for 2 patients with widely metastatic melanoma and non-small cell lung cancer. Model simulations suggest that only one or two tumor nodules in each patient serve as immunogenic hubs that could trigger an abscopal response. Radio-immunotherapy of any other lesion would result in local tumor control but systemic disease progression. Conclusions: Careful selection of immunogenic ‘hub’ nodules holds the key to successful abscopal disease control. The presented mathematical framework may help to identify the immunogenicity of each tumor nodule. Before serving as a novel decision support tool, rigorous calibration and validation remains to be pursued as more clinical abscopal data becomes available. Citation Format: Jan Poleszczuk, Sotiris Prokopiou, Mark Robertson-Tessi, Kimberly Luddy, Eduardo Moros, Mayer Fishman, Julie Djeu, Heiko Enderling. A systems biology approach to predict immunotherapy augmented abscopal effects. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A18.

112 Radiation-induced abscopal effect in normoxic and hypoxic conditions and hypoxia-induced abscopal effect in human lung cancer cells

112 Radiation-induced abscopal effect in normoxic and hypoxic conditions and hypoxia-induced abscopal effect in human lung cancer cells

A Case Report on Metastatic Thyroid Carcinoma: Radiation-induced Bystander or Abscopal Effect?

Purpose: Both anti-CD20 antibodies (ibritumomab; ZEVALIN® and tositumomab; BEXXAR®) currently used for radioimmunotherapy of B cell non-Hodgkin’s lymphoma are murine immunoglobulins. The aim of this feasibility study was to evaluate the safety and efficacy of radioimmunotherapy with a human chimeric anti-CD20 antibody labelled with Yttrium-90 (90Y-rituximab) in patients with B cell lymphoma. Methods: Patients with CD20+ B-cell lymphoma in partial remission or with progressive disease after at least one line of therapy were included. 90Y-rituximab was administered according to a similar schedule as currently approved by the European Medicines Agency for the treatment with 90Y-ibritumomab tiuxetan (ZEVALIN®): a first infusion of rituximab 250 mg/m² is repeated one week later and directly followed by the injection of 90Y-rituximab (14,8 MBq/kg). 18FDG-PET/CT was performed before treatment and repeated 3 months after for response assessment. Results: Twenty-six patients were treated with 90Y-rituximab. Disease histologies included mainly follicular lymphomas (53%). Toxicity was primarily haematological. The incidence of grade 3-4 neutropenia, thrombocytopenia and anemia were 34%, 38%, and 8% respectively, with spontaneous recovery in all but one patient that needed autologous stem cell transplant for refractory thrombocytopenia. Among the relevant long-term side effects, one patient developed secondary myelodysplasia 2 years after the treatment. The overall response rate was 88% (95% CI: 70%-98%), including 65% complete metabolic responses and 23% partial metabolic responses. After a median follow-up of 29.6 months, the Kaplan-Meier estimated median progression-free survival was 9, 1 months (95% CI 6,1-17,9). Median time to next treatment was 24 months (95% CI: 12, 2-28). Conclusion: Radioimmunotherapy with 90Y-rituximab in patients with relapsed CD20+ B-cell lymphomas is safe, well tolerated and effective when the ZEVALIN® treatment schedule is used.

Bystander-Type Effects Mediated by Long-Lived Inflammatory Signaling in Irradiated Bone Marrow

Radiation-induced bystander and abscopal effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate mechanisms of inducing damage and cell death additional to the conventional model of deposition of energy in the cell nucleus at the time of irradiation. In this study we show that signals generated in vivo in the bone marrow of mice irradiated with 4 Gy γ rays 18 h to 15 months previously are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells but that comparable signals are not detected at earlier times postirradiation or at doses below 100 mGy. Bone marrow cells of both CBA/Ca and C57BL/6 genotypes exhibit responses to signals produced by either irradiated CBA/Ca or C57BL/6 mice, and the responses are mediated by the cytokines FasL and TNF-α converging on a COX-2-dependent pathway. The findings are consistent with indirect inflammatory signaling induced as a response to the initial radiation damage rather than to direct signaling between irradiated and nonirradiated cells. The findings also demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.

Abstract 574: Mechanism of radiation-induced bystander effect: Role of NFκß pathway

Abstract Evidence from our lab and others clearly delineated that the transcriptional regulator NFκB plays a key role in induced responses in non-targeted bystander cells. We further reported curcumin impedes radiation (IR)-induced NFκB. However, full potential of curcumin is yet to be realized because of its poor bioavailability. This prompted us to investigate the efficacy of a more potent synthetic analogue of curcumin, EF24 in this setting. In this study, we investigated the orchestration of NFκB signaling after IR in an organ (lung) distant from the exposure field and elucidated the effect of EF24 in reverting the induced response. C57/BL6 mice either mock irradiated, exposed to single dose IR (SDR: 2 or 10Gy) or fractionated IR (FIR, 2 Gy/day for 5d) with or without EF24 (i.p. 200μg/Kg, 3h prior to IR) treatment were sacrificed after 24h. All IR exposures were limited to the lower abdomen area (1cm diameter), while the rest of the animal was protected by a specially designed cerrobend shield. EMSA analysis was used to examine the changes in NFκB DNA binding activity in lungs. Transcriptional activation of 88 NFκB pathway molecules was assessed using QPCR profiling. Phosphorylation of ERK1/2 and p38 that play a key role in NFκB signal transduction were examined using immunoblotting. DNA fragmentation was measured using TdT nick end labeling. A known NFκB dependent oxidative stress response SOD2 localization was examined using immunohistochemistry. IR significantly induced NFκB DNA binding activity in lungs. On the other hand, EF24 markedly suppressed IR-bystander effect (BSE)-induced NFκB DNA binding activity. QPCR profiling showed a significant and differential modulation in NFκB pathway transcriptome. To that end, of the 88 genes analyzed, 86, 86 and 70 genes were upregulated after 2Gy, 10Gy and FIR, respectively. Of these, 85, 80 and 63 genes were significantly (≫ 2 fold) upregulated. Conversely, EF24 completely suppressed 51, 52 and 43 SDR (2Gy, 10Gy) and FIR upregulated genes respectively in the non-targeted lungs. While IR profoundly phosphorylated ERK1/2 and p38 in distant lungs, EF24 treatment completely inhibited the phosphorylation of these kinases. Furthermore, IR-induced SOD2 levels in distant lung after all three IR-dose regimens were notably conferred by EF24 treatment. In addition, IR-enhanced DNA fragmentations in the lung tissue were significantly reduced with EF24. Taken together, these data clearly indicated that (i) an induced non-targeted effect occurs in mice lungs after clinically relevant doses of IR exposure; (ii) the orchestration of NFκB signal transduction may play a key role in induced abscopal responses; and (iii) EF24 treatment may attenuate NFκB and thereby alleviate radiation-induced abscopal effect at the distant organ. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 574. doi:10.1158/1538-7445.AM2011-574

Low LET Radiation-Induced Abscopal Effect and Tumor Recurrence: Nuclear Factor Kappa B (NF-κB) and Tumor Necrosis Factor alpha (TNF-α) Mediated Positive Feedback Mechanism.

Abstract The overall objective is to determine whether the surviving cancer cell after radiotherapy can initiate bystander response that result in clonal selection and tumor recurrence at the treatment site. We have two kinds of cell lines: human breast adenocarcinoma cells (MCF-7) and human aortic endothelism cells. First, in the MCF-7 cells we examined whether a bystander effect is initiated by TNF-a through activation of NF-kB which in-turn is triggered by therapeutic doses of radiation exposure, and also whether TNF-a and NF-kB can induce one another through positive feed back mechanisms and maintain a “memory” of the initial irradiation insult for an extended period. Second, between the MCF-7 and HAEC, we demonstrated whether radiation can induce the VEGF secreted by MCF-7, and concordant expression of the VEGFR in the bystander HAEC. MCF-7 cells were exposed to 137Cs γ ray to a total dose of 2 Gy at a dose rate of 1.06 Gy/min. Both NF-κB DNA-binding activity and the transcriptional activation measured by mobility shift assay and luciferase reporter assay, respectively showed radiation could induce nuclear translocation and transactivate κB-dependent gene expression. The activation was found to be bi-phasic, reached its maximum at 15 min (4.7-folds), decreased to basal level at 16 h, and reached a second phase maximum at 24 h (2.6-folds). Similarly, the levels of TNF-α determined by bioactivity assay revealed a 40% increase in soluble TNF-α in the culture supernatant upon 2 Gy exposure. Incubation of MCF-7 cells with either NF-κB inhibitor isohelenin or TNF-α neutralizing antibody blocked the presence of soluble TNF-α in the culture supernatant and second phase NF-κB activation, respectively. These results clearly indicated the occurrence of positive feed-back cycle between NF-κB and TNF-α through autocrine mechanism. In order to determine the occurrence of a similar mechanism in the non-irradiated bystander MCF-7 cells, supernatant from cells exposed to 2 Gy was added into the non-irradiated cells, and the NF-κB activity of non-irradiated MCF-7 cells increased by 7.2-folds. Next, to examine whether TNF-α is the paracrine signaling mediator in this bystander effect, un-irradiated MCF-7 cells, transiently transfected with NF-κB-luciferase reporter construct, was incubated with the conditioned medium from 2 Gy exposed cells in the presence or absence of TNF-α antibody. The luciferase activity showed a 60% increase in the un-irradiated MCF-7 cells when incubated with medium from irradiated cells in the absence of TNF-α antibody. Molecular analysis of specific angiogensis markers using hAngio-1 probe as a positive control was performed using ribonuclease protection assay. Of the angiogenic RNA molecules identified, the MCF-7 cells expressed VEGF and FGF-2, and the westernblot also showed the VEGF ligand and FGF-2 secreted by MCF-7 incresed by 3.4-folds and 2.6-folds separately after 2Gy exposure. We also co-cultured the irradiated MCF-7 cells with the non-irradiated HAEC, and found VEGFR2 and FGFR1 had corcondant expression increse in HAEC. This mechanism may be responsible for the stimulation of proliferative response, tumor-cell re-growth and subsequent tumor relapse at the treatment site. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2149.

The controversial abscopal effect

The abscopal effect is potentially important for tumor control and is mediated through cytokines and/or the immune system, mainly cell-mediated immunity. It results from loss of growth stimulatory and/or immunosuppressive factors from the tumor. Until recently, the abscopal effect referred to the distant effects seen after local radiation therapy. However, the term should now be used interchangeably with distant bystander effect. Through analysis of distant bystander effects of other local therapies, we discuss the poorly understood and researched radiation-induced abscopal effect. Although the abscopal effect has been described in various malignancies, it is a rarely recognized clinical event. The abscopal effect is still extremely controversial with known data that both support and refute the concept.