Radiation-Induced Bystander Effect: Loss of Radioprotective Capacity of Rosmarinic Acid In Vivo and In Vitro

In radiation oncology, the modulation of the bystander effect is a target both for the destruction of tumor cells and to protect healthy cells. With this objective, we determine whether the radioprotective capacity of rosmarinic acid (RA) can affect the intensity of these effects. Genoprotective capacity was obtained by determining the micronuclei frequencies in in vivo and in vitro assays and the cell survival was determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) (MTT) assay in three cell lines (PNT2, TRAMPC1 and B16F10), both in direct exposure to X-rays and after the production of radiation-induced bystander effect. The administration of RA in irradiated cells produced a decrease in the frequency of micronuclei both in vivo and in vitro, and an increase in cell survival, as expression of its radioprotective effect (p < 0.001) attributable to its ability to scavenge radio-induced free radicals (ROS). However, RA does not achieve any modification in the animals receiving serum or in the cultures treated with the irradiated medium, which expresses an absence of radioprotective capacity. The results suggest that ROS participates in the formation of signals in directly irradiated cells, but only certain subtypes of ROS, the cytotoxic products of lipid peroxidation, participate in the creation of lesions in recipient cells.

Oxidative Stress and Gene Expression Modifications Mediated by Extracellular Vesicles: An In Vivo Study of the Radiation-Induced Bystander Effect

Radiation-induced bystander effect is a biological response in nonirradiated cells receiving signals from cells exposed to ionising radiation. The aim of this in vivo study was to analyse whether extracellular vesicles (EVs) originating from irradiated mice could induce modifications in the redox status and expression of radiation-response genes in bystander mice. C57BL/6 mice were whole-body irradiated with 0.1-Gy and 2-Gy X-rays, and EVs originating from mice irradiated with the same doses were injected into naïve, bystander mice. Lipid peroxidation in the spleen and plasma reactive oxygen metabolite (ROM) levels increased 24 h after irradiation with 2 Gy. The expression of antioxidant enzyme genes and inducible nitric oxide synthase 2 (iNOS2) decreased, while cell cycle arrest-, senescence- and apoptosis-related genes were upregulated after irradiation with 2 Gy. In bystander mice, no significant alterations were observed in lipid peroxidation or in the expression of genes connected to cell cycle arrest, senescence and apoptosis. However, there was a systemic increase in the circulating ROM level after an intravenous EV injection, and EVs originating from 2-Gy-irradiated mice caused a reduced expression of antioxidant enzyme genes and iNOS2 in bystander mice. In conclusion, we showed that ionising radiation-induced alterations in the cellular antioxidant system can be transmitted in vivo in a bystander manner through EVs originating from directly irradiated animals.

Irradiated tumor cell–derived microparticles mediate tumor eradication via cell killing and immune reprogramming

Radiotherapy (RT) is routinely used in cancer treatment, but expansion of its clinical indications remains challenging. The mechanism underlying the radiation-induced bystander effect (RIBE) is not understood and not therapeutically exploited. We suggest that the RIBE is predominantly mediated by irradiated tumor cell–released microparticles (RT-MPs), which induce broad antitumor effects and cause immunogenic death mainly through ferroptosis. Using a mouse model of malignant pleural effusion (MPE), we demonstrated that RT-MPs polarized microenvironmental M2 tumor-associated macrophages (M2-TAMs) to M1-TAMs and modulated antitumor interactions between TAMs and tumor cells. Following internalization of RT-MPs, TAMs displayed increased programmed cell death ligand 1 (PD-L1) expression, enhancing follow-up combined anti–PD-1 therapy that confers an ablative effect against MPE and cisplatin-resistant MPE mouse models. Immunological memory effects were induced.

Surgical Wound Fluids from Patients with Breast Cancer Reveal Similarities in the Biological Response Induced by Intraoperative Radiation Therapy and the Radiation-Induced Bystander Effect—Transcriptomic Approach

In patients with breast cancer who undergo breast-conserving surgery (BCS), more than 90% of local recurrences occur in the same quadrant as the primary cancer. Surgical wound fluids (SWF) are believed to play a role in this process by inducing an inflammatory process in the scar tissue area. Despite strong clinical data demonstrating the benefits of intraoperative radiotherapy (IORT), the biological basis underlying this process remains poorly understood. Ionizing radiation (IR) directly affects cells by damaging DNA, thereby altering the cell phenotype. IR directly affects cancer cells and also influences unirradiated cells located nearby, a phenomenon known as the radiation-induced bystander effect (RIBE), significantly modifying the tumor microenvironment. We hypothesized that SWF obtained from patients after BCS and IORT would induce a radiobiological response (due to RIBE) in unirradiated cells, thereby modifying their phenotype. To confirm this hypothesis, breast cancer cells were incubated with SWF collected from patients after BCS: (1) without IORT (wound fluid (WF) group), (2) with IORT (radiotherapy wound fluid (RT-WF) group), and (3) WF with conditioned medium from irradiated cells (WF+RIBE group) and then subjected to microarray analysis. We performed gene set enrichment analysis to determine the biological processes present in these cells. This analysis showed that the RT-WF and WF+RIBE groups shared common biological processes, including the enhancement of processes involved in cell-cycle regulation, DNA repair, and oxidative phosphorylation. The WF group was characterized by overrepresentation of pathways involved in the INF-α and INF-γ response, inflammatory response, and the IL6 JAK/STAT3 signaling pathway. These findings show that MDA-MB-468 cells stimulated with surgical wound fluids obtained from patients who underwent BCS plus IORT and from cells stimulated with SWF plus RIBE share common biological processes. This confirms the role of the radiation-induced bystander effect in altering the biological properties of wound fluids.