Ionizing radiation effects on blood-derived extracellular vesicles: insights into miR-34a-5p-mediated cellular responses and biomarker potential.

Adverse effects of ionizing radiation on normal tissues limit the radiation dose in cancer treatment, thereby compromising treatment efficiency. Among the consistently affected non-cancer cells, peripheral blood mononuclear cells (PBMCs) exhibit high radiosensitivity and have the potential to induce systemic effects. PBMC-released extracellular vesicles (EVs), contribute to the communication of such systemic effects. This study aimed to investigate the effects of ionizing radiation on EVs as part of the systemic response of PBMCs in terms of microRNA cargo and biological functions.Therefore, whole blood samples from healthy donors were irradiated ex-vivo (0 Gy, 1 Gy, 2 Gy, 4 Gy) and EVs from PBMCs were isolated after 96 h by PEG precipitation or ultracentrifugation. Candidate microRNAs were examined in PBMC-derived EVs from individual donors. The uptake of membrane-stained fluorescent EVs by different recipient cells was quantified by fluorescence-activated cell sorting analysis. The biological effects of increased miR-34a-5p and of total EVs on recipient cells were assessed.Irradiation of PBMCs induced a dose-dependent upregulation of miR-34a-5p within EVs and PBMCs. However, interindividual differences between donors were noticed in the extent of upregulation, and small EVs displayed more pronounced changes in microRNA levels in comparison to large EVs. Irradiation in presence of the small molecule inhibitor KU-60019 demonstrated that this upregulation is dependent on ATM (Ataxia telangiectasia mutated) activation. Moreover, fibroblasts and keratinocytes were identified as preferred EV recipients. Increased miR-34a-5p levels led to a significant reduction in viability and induction of senescence in keratinocytes but not in fibroblasts, indicating a cell type-specific response.In conclusion, this study further elucidated the complex cellular response of normal tissue after radiation exposure. It confirmed radiation-induced modifications of microRNA expression levels in EVs from PBMCs and identified a robust upregulation of miR-34a-5p in the small EV subfraction, suggesting this microRNA as a potential novel candidate for the development of biomarkers for radiation exposure. Moreover, the different uptake efficiencies observed among specific cell types suggested that EVs induce cell type-specific responses in the intercellular communication of systemic radiation effects.

Radiomics approach for identifying radiation-induced normal tissue toxicity in the lung.

The rapidly evolving field of radiomics has shown that radiomic features are able to capture characteristics of both tumor and normal tissue that can be used to make accurate and clinically relevant predictions. In the present study we sought to determine if radiomic features can characterize the adverse effects caused by normal tissue injury as well as identify if human embryonic stem cell (hESC) derived extracellular vesicle (EV) treatment can resolve certain adverse complications. A cohort of 72 mice (n = 12 per treatment group) were exposed to X-ray radiation to the whole lung (3 × 8 Gy) or to the apex of the right lung (3 × 12 Gy), immediately followed by retro-orbital injection of EVs. Cone-Beam Computed Tomography images were acquired before and 2 weeks after treatment. In total, 851 radiomic features were extracted from the whole lungs and < 20 features were selected to train and validate a series of random forest classification models trained to predict radiation status, EV status and treatment group. It was found that all three classification models achieved significantly high prediction accuracies on a validation subset of the dataset (AUCs of 0.91, 0.86 and 0.80 respectively). In the locally irradiated lung, a significant difference between irradiated and unirradiated groups as well as an EV sparing effect were observed in several radiomic features that were not seen in the unirradiated lung (including wavelet-LLH Kurtosis, wavelet HLL Large Area High Gray Level Emphasis, and Gray Level Non-Uniformity). Additionally, a radiation difference was not observed in a secondary comparison cohort, but there was no impact of imaging machine parameters on the radiomic signature of unirradiated mice. Our data demonstrate that radiomics has the potential to identify radiation-induced lung injury and could be applied to predict therapeutic efficacy at early timepoints.

Biology of Exfoliation of Plasma Membrane-Derived Vesicles and the Radiation Response: Historical Background, Applications in Biodosimetry and Cell-Free Therapeutics, and Quantal Mechanisms for Their Release and Function with Implications for Space Travel.

This historical review of extracellular vesicles in the setting of exposure to ionizing radiation (IR) traces our understanding of how vesicles were initially examined and reported in the literature in the late 1970s (for secreted exosomes) and early 1980s (for plasma membrane-derived, exfoliated vesicles) to where we are now and where we may be headed in the next decade. An emphasis is placed on biophysical properties of extracellular vesicles, energy consumption and the role of vesiculation as an essential component of membrane turnover. The impact of intercellular signal trafficking by vesicle surface and intra-vesicular lipids, proteins, nucleic acids and metabolites is reviewed in the context of biomarkers for estimating individual radiation dose after exposure to radiation, pathogenesis of disease and development of cell-free therapeutics. Since vesicles express both growth stimulatory and inhibitory molecules, a hypothesis is proposed to consider superposition in a shared space and entanglement of molecules by energy sources that are external to human cells. Implications of this approach for travel in deep space are briefly discussed in the context of clinical disorders that have been observed after space travel.

Extracellular vesicles and the "six Rs" in radiotherapy.

Over half of patients with cancer receive radiation therapy during the course of their disease. Decades of radiobiological research have identified 6 parameters affecting the biological response to radiation referred to as the 6 "Rs": Repair, Radiosensitivity, Repopulation, Redistribution, Reoxygenation, and Reactivation of the anti-tumour immune response. Extracellular Vesicles (EVs) are small membrane-bound particles whose multiple biological functions are increasingly documented. Here we discuss the evidence for a role of EVs in the orchestration of the response of cancer cells to radiotherapy. We highlight that EVs are involved in DNA repair mechanisms, modulation of cellular sensitivity to radiation, and facilitation of tumour repopulation. Moreover, EVs influence tumour reoxygenation dynamics, and play a pivotal role in fostering radioresistance. Last, we examine how EV-related strategies could be translated into novel strategies aimed at enhancing the efficacy of radiation therapy against cancer.

Exploring the biphasic dose-response effects of photobiomodulation on the viability, migration, and extracellular vesicle secretion of human adipose mesenchymal stem cells.

Photobiomodulation (PBM) is a well-established medical technology that employs diverse light sources like lasers or light-emitting diodes to generate diverse photochemical and photophysical reactions in cells, thereby producing beneficial clinical outcomes. In this study, we introduced an 830 nm near-infrared (NIR) laser irradiation system combined with a microscope objective to precisely and controllably investigate the impact of PBM on the migration and viability of human adipose mesenchymal stem cells (hADSCs). We observed a biphasic dose-response in hADSCs' viability and migration after PBM exposure (0-10 J/cm2), with the 5 J/cm2 group showing significantly higher cell viability and migration ability than other groups. Additionally, at the optimal dose of 5 J/cm2, we used nanoparticle tracking analysis (NTA) and found a 6.25-fold increase in the concentration of extracellular vesicles (EVs) derived from hADSCs (PBM/ADSC-EVs) compared to untreated cells (ADSC-EVs). Both PBM/ADSC-EVs and ADSC-EVs remained the same size, with an average diameter of 56 nm measured by the ExoView R200 system, which falls within the typical size range for exosomes. These findings demonstrate that PBM not only improves the viability and migration of hADSCs but also significantly increases the EV yield.

The role and mechanism of engineered nanovesicles derived from hair follicle mesenchymal stem cells in the treatment of UVB-induced skin photoaging.

BACKGROUND: Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are effective in the treatment of skin photoaging; however, their low yield and functional decline with passage progression limit their clinical application. Cell-derived nanovesicles (CNVs) are potential alternatives that can address the limitations of EVs derived from MSCs and are conducive to clinical transformations. Hair follicle mesenchymal stem cells (HFMSCs), a type of MSCs, have demonstrated the function of repairing skin tissues; nevertheless, the efficacy of CNVs from HFMSCs (HFMSC-CNVs) in the treatment of skin photoaging remains unclear. Therefore, ultraviolet radiation B (UVB)-induced photoaging nude mice and human dermal fibroblasts (HDFs) were used as experimental models to investigate the therapeutic effects of HFMSC-CNVs in photoaging models. METHODS: HFMSC-CNVs were successfully prepared using the mechanical extrusion method. UVB-induced nude mice and HDFs were used as experimental models of photoaging. Multiple approaches, including hematoxylin-eosin and Masson staining, immunohistochemistry, immunofluorescence, detection of reactive oxygen species (ROS), flow cytometry, western blotting, and other experimental methods, were combined to investigate the possible effects and mechanisms of HFMSC-CNVs in the treatment of skin photoaging. RESULTS: In the nude mouse model of skin photoaging, treatment with HFMSC-CNVs reduced UVB-induced skin wrinkles (p < 0.05) and subcutaneous capillary dilation, alleviated epidermis thickening (p < 0.001), and dermal thinning (p < 0.001). Furthermore, HFMSC-CNVs upregulated proliferating cell nuclear antigen (PCNA) expression (p < 0.05) and decreased the levels of ROS, ß-galactosidase (ß-Gal), and CD86 (p < 0.01). In vitro experiments, treatment with HFMSC-CNVs enhanced the cellular activity of UVB-exposed HDFs (p < 0.05), and reduced ROS levels and the percentage of senescent cells (p < 0.001), and alleviated cell cycle arrest (p < 0.001). HFMSC-CNVs upregulated the expression of Collagen I (Col I), SMAD2/3, transforming growth factor beta (TGF-ß), catalase (CAT), glutathione peroxidase-1 (GPX-1), and superoxide dismutase-1 (SOD-1) (p < 0.05) and downregulated the expression of cycle suppressor protein (p53), cell cycle suppressor protein (p21), and matrix metalloproteinase 3 (MMP3) (p < 0.05). CONCLUSION: Conclusively, the anti-photoaging properties of HFMSC-CNVs were confirmed both in vivo and in vitro. HFMSC-CNVs exert anti-photoaging effects by alleviating cell cycle arrest, decreasing cellular senescence and macrophage infiltration, promoting cell proliferation and extracellular matrix (ECM) production, and reducing oxidative stress by increasing the activity of antioxidant enzymes.

Facilitatory effect of low-pulse repetition frequency ultrasound on release of extracellular vesicles from cultured myotubes.

PURPOSE: Extracellular vesicles (EVs) serve as carriers of intracellular factors with therapeutic effects, including tissue regeneration and attenuation of inflammatory responses. The majority of EVs in vivo are derived from skeletal muscle, which is reported to have anti-inflammatory effects. While high-intensity pulsed ultrasound (US) irradiation has been shown to promote EV secretion from myotubes, the impact of pulse repetition frequency, a US parameter affecting pulse length, on EV release remains unclear. This study aimed to investigate the impact of pulse repetition frequency of US on the release of EVs from myotubes. METHODS: C2C12 myoblasts were used in this study. After differentiation into C2C12 myotubes, US was performed for 5 min at an intensity of 3.0 W/cm2, duty cycle of 20%, acoustic frequency of 1 MHz, and different pulse repetition frequencies (100 Hz, 10 Hz, or 1 Hz). After 12 h, EVs and cells were collected for subsequent analyses. RESULTS: US did not cause a reduction in cell viability across all US groups compared to the control. The concentration of EVs was significantly higher in all US groups compared to the control group. In particular, the highest increase was observed in the 1-Hz group on EV concentration as well as intracellular Ca2+ level. CONCLUSION: This study investigated the effect of three different pulse repetition frequencies of US on the release of EVs from cultured myotubes. It is concluded that a low-pulse repetition frequency of 1 Hz is the most effective for enhancing EV release from cultured myotubes with pulsed ultrasound.

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy.

PURPOSE: Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil 7Li, which have a path length of 5-9 µm, are generated by the capture reaction between 10B and thermal neutrons and selectively kill tumor cells that have uptaken 10B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. METHOD: Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO2 incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. RESULT: An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. CONCLUSION: These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma.

Extracellular vesicles mediated proinflammatory macrophage phenotype induced by radiotherapy in cervical cancer.

BACKGROUND: Radiotherapy is a highly effective treatment for cervical cancer. Recent studies focused on the radiotherapy induced anti-tumor immunity. Whether tumor-derived extracellular vesicles (EVs) play roles in radiotherapy induced tumor associated macrophage (TAM) polarization remains unclear. MATERIALS AND METHODS: This study analysed the phenotype of macrophages in cancer tissue and peripheral blood of cervical cancer patients using flow cytometry analysis. The role of EVs from plasma of post-irradiated patients on M2-like transformed macrophages was assessed. The M1- and M2-like macrophages were assessed by expression of cell surface markers (CCR7, CD163) and intracellular cytokines (IL-10, TNFα and iNOS). The capacity of phagocytosis was assessed by PD-1 expression and phagocytosis of pHrodo Red E. coli bioparticles. RESULTS: Our results demonstrated that radiotherapy of cervical cancer induced an increase in the number of TAMs and a change in their subtype from the M2-like to the M1-like phenotype (increased expression of CCR7 and decreased expression of CD163). The EVs from plasma of post-irradiated patients facilitated the M2-like to the M1-like phenotype transition (increased expression of CCR7, TNFα and iNOS, and decreased expression of CD163 and IL-10) and increased capacity of phagocytosis (decreased PD-1 expression and increased phagocytosis of pHrodo Red E. coli bioparticles). CONCLUSIONS: Our data demonstrated that irradiation in cervical cancer patients facilitated a proinflammatory macrophage phenotype which could eventually able to mediate anti-tumor immune responses. Our findings highlight the importance of EV in the crosstalk of tumor cells and TAM upon irradiation, which potentially leading to an increased inflammatory response to cancer lesions.

Dental pulp stem cell-derived small extracellular vesicle in irradiation-induced senescence.

Small extracellular vesicles (sEV) facilitate signaling molecule transfer among cells. We examined the therapeutic efficacy of human dental pulp stem cell-derived sEV (hDPSC-sEV) against cellular senescence in an irradiated-submandibular gland mouse model. Seven-week-old mice were exposed to 25 Gy radiation and randomly assigned to control, phosphate-buffered saline (PBS), or hDPSC-sEV groups. At 18 days post-irradiation, saliva production was measured; histological and reverse transcription-quantitative PCR analyses of the submandibular glands were performed. The salivary flow rate did not differ significantly between the PBS and hDPSC-sEV groups. AQP5-expressing acinar cell numbers and AQP5 expression levels in the submandibular glands were higher in the hDPSC-sEV group than in the other groups. Furthermore, compared with non-irradiated mice, mice in the 25 Gy + PBS group showed a high senescence-associated-ß-galactosidase-positive cell number and upregulated senescence-related gene (p16INK4a, p19Arf, p21) and senescence-associated secretory phenotypic factor (MMP3, IL-6, PAI-1, NF-κB, and TGF-ß) expression, all of which were downregulated in the hDPSC-sEV group. Superoxide dismutase levels were lower in the PBS group than in the hDPSC-sEV group. In summary, hDPSC-sEV reduced inflammatory cytokine and senescence-related gene expression and reversed oxidative stress in submandibular cells, thereby preventing irradiation-induced cellular senescence. Based on these results, we hope to contribute to the development of innovative treatment methods for salivary gland dysfunction that develops after radiotherapy for head and neck cancer.

Secretion rates and protein composition of extracellular vesicles released by cancer-associated fibroblasts after radiation.

Reciprocal communication between the malignant and non-malignant cellular elements in tumors is essential for cancer sustainability and plays an important role in the response of cancers to treatments. Some of this cellular crosstalk takes place via secretion of vesicles that are actively released into the extracellular space by most cell types in tumors. Recent studies have demonstrated radiation-induced changes in the secretion rate and composition of extracellular vesicles (EVs), with impact on radiation-related cellular communication. However, little is known about the effects of different radiation regimens on the release of EVs by cells of the tumor microenvironment. In this study, we provide a comprehensive molecular characterization of EVs released by cultured primary lung tumor fibroblasts. We explore the quantitative and morphological changes triggered by ionizing radiation (IR), delivered as a single dose of 18 Gy or three consecutive daily medium-doses of 6 Gy. Cancer-associated fibroblasts (CAFs) secrete EVs with sizes ranging from 80 to 200 nm, expressing some of the classical exosome markers. Exposing CAFs to a single-high radiation dose (1 × 18 Gy) or fractionated medium-dose did not alter the release of CAF-EVs. The protein composition of CAF-EVs was analyzed by LC-MS/MS proteomics and revealed that CAF-EVs are enriched with heat shock proteins, integrins, tetraspanins, proteinases, collagens, growth factors and an array of molecules involved in the regulation of cell migration and the immune system. Quantitative proteomic analyses revealed minor changes in the protein composition of CAF-EVs after radiation exposure. Taken together, this study presents original data on lung tumor CAF-EV composition and reveals that release and protein cargo of CAF-EVs are largely unaltered after exposing CAFs to IR.

Characterization of exosome release and extracellular vesicle-associated miRNAs for human bronchial epithelial cells irradiated with high charge and energy ions.

Exosomes are extracellular vesicles that mediate transport of nucleic acids, proteins, and other molecules. Prior work has implicated exosomes in the transmission of radiation nontargeted effects. Here we investigate the ability of energetic heavy ions, representative of species found in galactic cosmic rays, to stimulate exosome release from human bronchial epithelial cells in vitro. Immortalized human bronchial epithelial cells (HBEC3-KT F25F) were irradiated with 1.0 Gy of high linear energy transfer (LET) 48Ti, 28Si, or 16O ions, or with 10 Gy of low-LET reference γ-rays, and extracellular vesicles were collected from conditioned media. Preparations were characterized by single particle tracking analysis, transmission electron microscopy, and immunoblotting for the exosomal marker, TSG101. Based on TSG101 levels, irradiation with high-LET ions, but not γ-rays, stimulated exosome release by about 4-fold, relative to mock-irradiated controls. The exosome-enriched vesicle preparations contained pro-inflammatory damage-associated molecular patterns, including HSP70 and calreticulin. Additionally, miRNA profiling was performed for vesicular RNAs using NanoString technology. The miRNA profile was skewed toward a small number of species that have previously been shown to be involved in cancer initiation and progression, including miR-1246, miR-1290, miR-23a, and miR-205. Additionally, a set of 24 miRNAs was defined as modestly over-represented in preparations from HZE ion-irradiated versus other cells. Gene set enrichment analysis based on the over-represented miRNAs showed highly significant association with nonsmall cell lung and other cancers.

Ionizing Radiation-Induced Extracellular Vesicle Release Promotes AKT-Associated Survival Response in SH-SY5Y Neuroblastoma Cells.

Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms of neuroblastoma, radiation can increase the risk of secondary neoplasms, due to the ability of irradiated cells to transmit pro-survival signals to non-irradiated cells through vesicle secretion. The aims of this study were to characterize the vesicles released by the human neuroblastoma cell line SH-SY5Y following X-ray radiations and their ability to increase invasiveness in non-irradiated SH-SY5Y cells. We first purified the extracellular vesicles released by the SH-SY5Y cells following X-rays, and then determined their total amount, dimensions, membrane protein composition, and cellular uptake. We also examined the effects of these extracellular vesicles on viability, migration, and DNA damage in recipient SH-SY5Y cells. We found that exposure to X-rays increased the release of extracellular vesicles and altered their protein composition. These vesicles were readily uptaken by non-irradiated cells, inducing an increase in viability, migration, and radio-resistance. The same results were obtained in an MYCN-amplified SK-N-BE cell line. Our study demonstrates that vesicles released from irradiated neuroblastoma cells stimulate proliferation and invasiveness that correlate with the epithelial to mesenchymal transition in non-irradiated cells. Moreover, our results suggest that, at least in neuroblastomas, targeting the extracellular vesicles may represent a novel therapeutic approach to counteract the side effects associated with radiotherapy.

Extracellular vesicles released from irradiated neonatal mouse cheek tissue increased cell survival after radiation.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance.

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC.

The effect of ionising radiation on the phenotype of bone marrow-derived extracellular vesicles.

OBJECTIVES: Ionising radiation-induced alterations affecting intercellular communication in the bone marrow (BM) contribute to the development of haematological pathologies. Extracellular vesicles (EVs), which are membrane-coated particles released by cells, have important roles in intercellular signalling in the BM. Our objective was to investigate the effects of ionising radiation on the phenotype of BM-derived EVs of total-body irradiated mice. METHODS: CBA mice were irradiated with 0.1 Gy or 3 Gy X-rays. BM was isolated from the femur and tibia 24 h after irradiation. EVs were isolated from the BM supernatant. The phenotype of BM cells and EVs was analysed by flow cytometry. RESULTS: The mean size of BM-derived EVs was below 300 nm and was not altered by ionising radiation. Their phenotype was very heterogeneous with EVs carrying either CD29 or CD44 integrins representing the major fraction. High-dose ionising radiation induced a strong rearrangement in the pool of BM-derived EVs which were markedly different from BM cell pool changes. The proportion of CD29 and CD44 integrin-harbouring EVs significantly decreased and the relative proportion of EVs with haematopoietic stem cell or lymphoid progenitor markers increased. Low-dose irradiation had limited effect on EV secretion. CONCLUSIONS: Ionising radiation induced selective changes in the secretion of EVs by the different BM cell subpopulations. ADVANCES IN KNOWLEDGE: The novelty of the paper consists of performing a detailed phenotyping of BM-derived EVs after in vivo irradiation of mice.

Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity.

Background: Increasing evidence points to the critical role of extracellular vesicles (EVs) as molecular parcels that carry a diverse array of bioactive payloads for coordination of complex intracellular signaling. Focused ultrasound (FUS) hyperthermia is a technique for non-invasive, non-ionizing sublethal heating of cells in a near-instantaneous manner; while it has been shown to improve drug delivery and immunological recognition of tumors, its impact on EVs has not been explored to date. The goal of this study was to determine whether FUS impacts the release, proteomic profile, and immune-activating properties of tumor-derived EVs. Methods: Monolayered murine glioma cells were seeded within acoustically transparent cell culture chambers, and FUS hyperthermia was applied to achieve complete coverage of the chamber. Glioma-derived EVs (GEVs) were isolated for characterization by Nanoparticle Tracking Analysis, cryo-electron microscopy and mass spectrometry. An in vitro experimental setup was designed to further dissect the impact of GEVs on innate inflammation; immortalized murine dendritic cells (DCs) were pulsed with GEVs (either naïve or FUS hyperthermia-exposed) and assayed for production of IL-12p70, an important regulator of DC maturation and T helper cell polarization toward the interferon-γ-producing type 1 phenotype. Results: We confirmed that FUS hyperthermia significantly augments GEV release (by ~46%) as well as shifts the proteomic profile of these GEVs. Such shifts included enrichment of common EV-associated markers, downregulation of markers associated with cancer progression and resistance and modulation of inflammation-associated markers. When DCs were pulsed with GEVs, we noted that naïve GEVs suppressed IL-12p70 production by DCs in a GEV dose-dependent manner. In contrast, GEVs from cells exposed to FUS hyperthermia promoted a significant upregulation in IL-12p70 production by DCs, consistent with a pro-inflammatory stimulus. Conclusion: FUS hyperthermia triggers release of proteomically distinct GEVs that are capable of facilitating an important component of innate immune activation, lending both to a potential mechanism by which FUS interfaces with the tumor-immune landscape and to a role for GEV-associated biomarkers in monitoring response to FUS.

Radiation-induced extracellular vesicle (EV) release of miR-603 promotes IGF1-mediated stem cell state in glioblastomas.

BACKGROUND: Recurrence after radiation therapy is nearly universal for glioblastomas, the most common form of adult brain cancer. The study aims to define clinically pertinent mechanisms underlying this recurrence. METHODS: microRNA (miRNA) profiling was performed using matched pre- and post-radiation treatment glioblastoma specimens from the same patients. All specimens harbored unmethylated O6-methylguanine-DNA methyltransferase promoters (umMGMT) and wild-type isocitrate dehydrogenase (wtIDH). The most altered miRNA, miR-603, was characterized. FINDINGS: While nearly all miRNAs remained unchanged after treatment, decreased levels of few, select miRNAs in the post-treatment specimens were observed, the most notable of which involved miR-603. Unbiased profiling of miR-603 targets revealed insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). Ionizing radiation (IR) induced cellular export of miR-603 through extracellular vesicle (EV) release, thereby de-repressing IGF1 and IGF1R. This de-repression, in turn, promoted cancer stem-cell (CSC) state and acquired radiation resistance in glioblastomas. Export of miR-603 additionally de-repressed MGMT, a DNA repair protein responsible for detoxifying DNA alkylating agents, to promote cross-resistance to these agents. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and DNA alkylating agents. INTERPRETATION: Profiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. FUNDING: NIH 1R01NS097649-01, 9R44GM128223-02, 1R01CA240953-01, the Doris Duke Charitable Foundation Clinical Scientist Development Award, The Sontag Foundation Distinguished Scientist Award, the Kimmel Scholar Award, and BWF 1006774.01 (C.C.C).

Radiation-induced augmentation in dendritic cell function is mediated by apoptotic bodies/STAT5/Zbtb46 signaling.

Purpose: To evaluate the effect of ionizing radiation (IR) exposure on differentiation and maturation of dendritic cells (DC).Materials and methods: Bone marrow progenitor cells irradiated in vitro or isolated from mice exposed to whole body or localized tumor irradiation were differentiated into DC. Phenotypic maturation of DC was characterized by labeling with specific antibodies and flow cytometry analysis. Cytokines were estimated by ELISA.Results: Splenic and bone marrow-derived DC (BMDC) from tumor-bearing mice exposed to localized irradiation showed abrogation of tumor-induced immunosuppression. This was not due to the effect of radiation on tumor cells as DC derived from normal mice exposed to whole-body irradiation (WBI) also showed increase in immune-activating potential of DC. This was observed in terms of increased phenotypic and functional activation of DCs. This phenomenon was also recapitulated if DC were differentiated from in vitro irradiated progenitor cells and was found to be due to STAT5/Zbtb46 signaling mediated by the irradiation-induced apoptotic bodies (ABs). When these ABs were depleted using annexin-beads, these effects were reversed confirming the involvement of this pathway. The role of ABs was further validated in DC derived from mice exposed to WBI in adaptive response experiments with 0.1 Gy priming dose prior to 2 Gy challenge dose. A corresponding reduction in DC maturation markers was observed with decrease in apoptosis in vivo. Further, these DCs derived from irradiated progenitors (IP) could resist the suppressive effects of tumor conditioned medium (TCM) and had increased immune-activating potential as seen in the tumor-bearing mice.Conclusions: Though radiation is the most commonly used therapeutic modality for cancer, its effects on dendritic cell differentiation is not completely understood. We demonstrate here for the first time that exposure to select doses of IR can increase immune-activating potential of DC through ABs. This can have implications in selection of appropriate doses of IR during radiotherapy of cancer patients.

Radiation-induced small extracellular vesicles as "carriages" promote tumor antigen release and trigger antitumor immunity.

Rationale: Accumulating evidence supports the importance of radiation therapy in the induction of antitumor immunity. Small extracellular vesicles (sEVs) play essential roles in tumor antigen loading and delivery. However, the role of sEVs in radiation-induced antitumor immunity remains unclear. It is therefore important to determine the role and regulatory mechanisms of sEVs in radiation-induced immunity. Methods: Tumor cells were irradiated (8 Gy), and sEVs were purified via ultracentrifugation. Primary tumor and experimental lung metastasis models were established in mice to evaluate antitumor immunity triggered by immunization with sEVs. Proteomic and bioinformatic analyses were performed to identify altered cargos in sEVs induced by radiation. Peptides derived from up-regulated proteins in sEVs were designed and synthesized as vaccines according to major histocompatibility complex (MHC) I binding and immunogenicity. Results: Here, we demonstrated that sEVs derived from irradiated tumor cells could trigger antitumor immunity against primary tumor and experimental lung metastasis by enhancing CD8+ and CD4+ T cell infiltration. Radiation may also enrich sEVs with tumor antigens and heat-shock proteins. Furthermore, CUB domain-containing protein 1 (CDCP1) derived from radiation-induced sEVs was identified as a novel tumor-associated antigen and developed as a peptide vaccine that may generate antitumor immune responses. Conclusions: Our results demonstrate that the use of sEVs secreted by irradiated tumor cells constitutes an efficient approach for tumor antigen delivery and presentation and highlight the role of sEVs in radiation-triggered antitumor immunity.