Extracellular Vesicles Derived from Bone Marrow in an Early Stage of Ionizing Radiation Damage Are Able to Induce Bystander Responses in the Bone Marrow

Ionizing radiation (IR)-induced bystander effects contribute to biological responses to radiation, and extracellular vesicles (EVs) play important roles in mediating these effects. In this study we investigated the role of bone marrow (BM)-derived EVs in the bystander transfer of radiation damage. Mice were irradiated with 0.1Gy, 0.25Gy and 2Gy, EVs were extracted from the BM supernatant 24 h or 3 months after irradiation and injected into bystander mice. Acute effects on directly irradiated or EV-treated mice were investigated after 4 and 24 h, while late effects were investigated 3 months after treatment. The acute effects of EVs on the hematopoietic stem and progenitor cell pools were similar to direct irradiation effects and persisted for up to 3 months, with the hematopoietic stem cells showing the strongest bystander responses. EVs isolated 3 months after irradiation elicited no bystander responses. The level of seven microRNAs (miR-33a-3p, miR-140-3p, miR-152-3p, miR-199a-5p, miR-200c-5p, miR-375-3p and miR-669o-5p) was altered in the EVs isolated 24 hour but not 3 months after irradiation. They regulated pathways highly relevant for the cellular response to IR, indicating their role in EV-mediated bystander responses. In conclusion, we showed that only EVs from an early stage of radiation damage could transmit IR-induced bystander effects.

A general theoretical framework to design base editors with reduced bystander effects

Base editors (BEs) hold great potential for medical applications of gene therapy. However, high precision base editing requires BEs that can discriminate between the target base and multiple bystander bases within a narrow active window (4 – 10 nucleotides). Here, to assist in the design of these optimized editors, we propose a discrete-state stochastic approach to build an analytical model that explicitly evaluates the probabilities of editing the target base and bystanders. Combined with all-atom molecular dynamic simulations, our model reproduces the experimental data of A3A-BE3 and its variants for targeting the “TC” motif and bystander editing. Analyzing this approach, we propose several general principles that can guide the design of BEs with a reduced bystander effect. These principles are then applied to design a series of point mutations at T218 position of A3G-BEs to further reduce its bystander editing. We verify experimentally that the new mutations provide different levels of stringency on reducing the bystander editing at different genomic loci, which is consistent with our theoretical model. Thus, our study provides a computational-aided platform to assist in the scientifically-based design of BEs with reduced bystander effects.

Revisiting the Radiobiology of Targeted Alpha Therapy

Targeted alpha therapy (TAT) using alpha particle-emitting radionuclides is in the spotlight after the approval of 223RaCl2 for patients with metastatic castration-resistant prostate cancer and the development of several alpha emitter-based radiopharmaceuticals. It is acknowledged that alpha particles are highly cytotoxic because they produce complex DNA lesions. Hence, the nucleus is considered their critical target, and many studies did not report any effect in other subcellular compartments. Moreover, their physical features, including their range in tissues (<100 μm) and their linear energy transfer (50–230 keV/μm), are well-characterized. Theoretically, TAT is indicated for very small-volume, disseminated tumors (e.g., micrometastases, circulating tumor cells). Moreover, due to their high cytotoxicity, alpha particles should be preferred to beta particles and X-rays to overcome radiation resistance. However, clinical studies showed that TAT might be efficient also in quite large tumors, and biological effects have been observed also away from irradiated cells. These distant effects are called bystander effects when occurring at short distance (<1 mm), and systemic effects when occurring at much longer distance. Systemic effects implicate the immune system. These findings showed that cells can die without receiving any radiation dose, and that a more complex and integrated view of radiobiology is required. This includes the notion that the direct, bystander and systemic responses cannot be dissociated because DNA damage is intimately linked to bystander effects and immune response. Here, we provide a brief overview of the paradigms that need to be revisited.

A Proteomic Study Suggests Stress Granules as New Potential Actors in Radiation-Induced Bystander Effects

Besides the direct effects of radiations, indirect effects are observed within the surrounding non-irradiated area; irradiated cells relay stress signals in this close proximity, inducing the so-called radiation-induced bystander effect. These signals received by neighboring unirradiated cells induce specific responses similar with those of direct irradiated cells. To understand the cellular response of bystander cells, we performed a 2D gel-based proteomic study of the chondrocytes receiving the conditioned medium of low-dose irradiated chondrosarcoma cells. The conditioned medium was directly analyzed by mass spectrometry in order to identify candidate bystander factors involved in the signal transmission. The proteomic analysis of the bystander chondrocytes highlighted 20 proteins spots that were significantly modified at low dose, implicating several cellular mechanisms, such as oxidative stress responses, cellular motility, and exosomes pathways. In addition, the secretomic analysis revealed that the abundance of 40 proteins in the conditioned medium of 0.1 Gy irradiated chondrosarcoma cells was significantly modified, as compared with the conditioned medium of non-irradiated cells. A large cluster of proteins involved in stress granules and several proteins involved in the cellular response to DNA damage stimuli were increased in the 0.1 Gy condition. Several of these candidates and cellular mechanisms were confirmed by functional analysis, such as 8-oxodG quantification, western blot, and wound-healing migration tests. Taken together, these results shed new lights on the complexity of the radiation-induced bystander effects and the large variety of the cellular and molecular mechanisms involved, including the identification of a new potential actor, namely the stress granules.