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 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 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 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.

Extracellular vesicles originating from glioblastoma cells increase metalloproteinase release by astrocytes: the role of CD147 (EMMPRIN) and ionizing radiation.

BACKGROUND: Glioblastoma multiforme is an aggressive primary brain tumor that is characterized by local invasive growth and resistance to therapy. The role of the microenvironment in glioblastoma invasiveness remains unclear. While carcinomas release CD147, a protein that signals for increased matrix metalloproteinase (MMP) release by fibroblasts, glioblastoma does not have a significant fibroblast component. We hypothesized that astrocytes release MMPs in response to CD147 contained in glioblastoma-derived extracellular vesicles (EVs) and that ionizing radiation, part of the standard treatment for glioblastoma, enhances this release. METHODS: Astrocytes were incubated with EVs released by irradiated or non-irradiated human glioblastoma cells wild-type, knockdown, or knockout for CD147. Levels of CD147 in glioblastoma EVs and MMPs secreted by astrocytes were quantified. Levels of proteins in the mitogen activated protein kinase (MAPK) pathway, which can be regulated by CD147, were measured in astrocytes incubated with EVs from glioblastoma cells wild-type or knockdown for CD147. Immunofluorescence was performed on the glioblastoma cells to identify changes in CD147 localization in response to irradiation, and to confirm uptake of the EVs by astrocytes. RESULTS: Immunoblotting and mass spectrometry analyses showed that CD147 levels in EVs were transiently increased when the EVs were from glioblastoma cells that were irradiated with γ rays. Specifically, the highly-glycosylated 45 kDa form of CD147 was preferentially present in the EVs relative to the cells themselves. Immunofluorescence demonstrated that astrocytes incorporate glioblastoma EVs and subsequently increase their secretion of active MMP9. The increase was greater if the EVs were from irradiated glioblastoma cells. Testing MAPK pathway activation, which also regulates MMP expression, showed that JNK signaling, but not ERK1/2 or p38, was increased in astrocytes incubated with EVs from irradiated compared to non-irradiated glioblastoma cells. Knockout of CD147 in glioblastoma cells blocked the increased JNK signaling and the rise in secreted active MMP9 levels. CONCLUSIONS: The results support a tumor microenvironment-mediated role of CD147 in glioblastoma invasiveness, and reveal a prominent role for ionizing radiation in enhancing the effect. They provide an improved understanding of glioblastoma intercellular signaling in the context of radiotherapy, and identify pathways that can be targeted to reduce tumor invasiveness. Video abstract.

Low-intensity pulsed ultrasound affects growth, differentiation, migration, and epithelial-to-mesenchymal transition of colorectal cancer cells.

Ultrasound (US) offers potentially important opportunities from a therapeutic point of view. Thus, the study of the biological effects of US on cancer cells is important to understand the consequences of these changes on the malignant phenotype. This study aimed to investigate the effects of low-intensity ultrasound (LIPUS) on the phenotype of colorectal cancer cell lines. Cell proliferation was evaluated by viability test and by evaluation of pERK expression, while cell motility using the scratch test. Cell differentiation was evaluated assessing alkaline phosphatase activity. Epithelial mesenchymal transition was assessed by analyzing the expression of Vimentin and E-Cadherin. Release and uptake of extracellular vesicles (EVs) were evaluated by flow cytometry. LIPUS effects on the organization of cytoskeleton were analyzed by confocal microscopy and by evaluation of Rho GTPase expression. No alterations in vitality and clonogenicity were observed when the intermediate (0.4 MPa) and the lowest (0.035 MPa) acoustic intensities were administered while the treatment with high intensity (1 MPa) induced a reduction of both cell viability and clonogenicity in both cell lines in a frequency-dependent manner. LIPUS promoted the differentiation of colon cancer cells, affected epithelial-to-mesenchymal transition, promoted the closure of a wound as well as increased the release of EVs compared with untreated cells. LIPUS-induced increase in cell motility was likely due to a Rho GTPase-dependent mechanism. Overall, the results obtained warrant further studies on the potential combined effect of LIPUS with differentiating agents and on their potential use in a clinical setting.

Extracellular vesicles released by melanocytes after UVA irradiation promote intercellular signaling via miR21.

Skin pigmentation is controlled by complex crosstalk between melanocytes and keratinocytes and is primarily induced by exposure to ultraviolet (UV) irradiation. Several aspects of UVA-induced signaling remain to be explored. In skin cells, UVA induces plasma membrane damage, which is repaired by lysosomal exocytosis followed by instant shedding of extracellular vesicles (EVs) from the plasma membrane. The released EVs are taken up by neighboring cells. To elucidate the intercellular crosstalk induced by UVA irradiation, EVs were purified from UVA-exposed melanocytes and added to keratinocytes. Transcriptome analysis of the keratinocytes revealed the activation of TGF-ß and IL-6/STAT3 signaling pathways and subsequent upregulation of microRNA (miR)21. EVs induced phosphorylation of ERK and JNK, reduced protein levels of PDCD4 and PTEN, and augment antiapoptotic signaling. Consequently, keratinocyte proliferation and migration were stimulated and UV-induced apoptosis was significantly reduced. Interestingly, melanoma cells and melanoma spheroids also generate increased amounts of EVs with capacity to stimulate proliferation and migration upon UVA. In conclusion, we present a novel intercellular crosstalk mediated by UVA-induced lysosome-derived EVs leading to the activation of proliferation and antiapoptotic signaling via miR21.

Extracellular Vesicles in Modifying the Effects of Ionizing Radiation.

Extracellular vesicles (EVs) are membrane-coated nanovesicles actively secreted by almost all cell types. EVs can travel long distances within the body, being finally taken up by the target cells, transferring information from one cell to another, thus influencing their behavior. The cargo of EVs comprises of nucleic acids, lipids, and proteins derived from the cell of origin, thereby it is cell-type specific; moreover, it differs between diseased and normal cells. Several studies have shown that EVs have a role in tumor formation and prognosis. It was also demonstrated that ionizing radiation can alter the cargo of EVs. EVs, in turn can modulate radiation responses and they play a role in radiation-induced bystander effects. Due to their biocompatibility and selective targeting, EVs are suitable nanocarrier candidates of drugs in various diseases, including cancer. Furthermore, the cargo of EVs can be engineered, and in this way they can be designed to carry certain genes or even drugs, similar to synthetic nanoparticles. In this review, we describe the biological characteristics of EVs, focusing on the recent efforts to use EVs as nanocarriers in oncology, the effects of EVs in radiation therapy, highlighting the possibilities to use EVs as nanocarriers to modulate radiation effects in clinical applications.

Plasma-derived extracellular vesicles yield predictive markers of cranial irradiation exposure in mice.

Ionizing radiation exposure to the brain is common for patients with a variety of CNS related malignancies. This exposure is known to induce structural and functional alterations to the brain, impacting dendritic complexity, spine density and inflammation. Over time, these changes are associated with cognitive decline. However, many of these impacts are only observable long after irradiation. Extracellular vesicles (EVs) are shed from cells in nearly all known tissues, with roles in many disease pathologies. EVs are becoming an important target for identifying circulating biomarkers. The aim of this study is to identify minimally invasive biomarkers of ionizing radiation damage to the CNS that are predictors of late responses that manifest as persistent cognitive impairments. Using a clinically relevant 9 Gy irradiation paradigm, we exposed mice to cranial (head only) irradiation. Using metabolomic and lipidomic profiling, we analyzed their plasma and plasma-derived EVs two days and two weeks post-exposure to detect systemic signs of damage. We identified significant changes associated with inflammation in EVs. Whole-plasma profiling provided further evidence of systemic injury. These studies are the first to demonstrate that profiling of plasma-derived EVs may be used to study clinically relevant markers of ionizing radiation toxicities to the brain.

Ionizing radiation affects the composition of the proteome of extracellular vesicles released by head-and-neck cancer cells in vitro.

Exosomes and other extracellular vesicles are key players in cell-to-cell communication, and it has been proposed that they are involved in different aspects of the response to ionizing radiation, including transmitting the radiation-induced bystander effect and mediating radioresistance. The functional role of exosomes depends on their molecular cargo, including proteome content. Here we aimed to establish the proteome profile of exosomes released in vitro by irradiated UM-SCC6 cells derived from human head-and-neck cancer and to identify processes associated with radiation-affected proteins. Exosomes and other small extracellular vesicles were purified by size-exclusion chromatography from cell culture media collected 24 h after irradiation of cells with a single 2, 4 or 8 Gy dose, and then proteins were identified using a shotgun LC-MS/MS approach. Exosome-specific proteins encoded by 1217 unique genes were identified. There were 472 proteins whose abundance in exosomes was significantly affected by radiation (at any dose), including 425 upregulated and 47 downregulated species. The largest group of proteins affected by radiation (369 species) included those with increased abundance at all radiation doses (≥2 Gy). Several gene ontology terms were associated with radiation-affected exosome proteins. Among overrepresented processes were those involved in the response to radiation, the metabolism of radical oxygen species, DNA repair, chromatin packaging, and protein folding. Hence, the protein content of exosomes released by irradiated cells indicates their actual role in mediating the response to ionizing radiation.

Extracellular vesicles mediate low dose ionizing radiation-induced immune and inflammatory responses in the blood.

PURPOSE: Radiation-induced bystander effects (RIBE) imply the involvement of complex signaling mechanisms, which can be mediated by extracellular vesicles (EVs). Using an in vivo model, we investigated EV-transmitted RIBE in blood plasma and radiation effects on plasma EV miRNA profiles. MATERIALS AND METHODS: C57Bl/6 mice were total-body irradiated with 0.1 and 2 Gy, bone marrow-derived EVs were isolated, and injected systemically into naive, 'bystander' animals. Proteome profiler antibody array membranes were used to detect alterations in plasma, both in directly irradiated and bystander mice. MiRNA profile of plasma EVs was determined by PCR array. RESULTS: M-CSF and pentraxin-3 levels were increased in the blood of directly irradiated and bystander mice both after low and high dose irradiations, CXCL16 and lipocalin-2 increased after 2 Gy in directly irradiated and bystander mice, CCL5 and CCL11 changed in bystander mice only. Substantial overlap was found in the cellular pathways regulated by those miRNAs whose level were altered in EVs isolated from the plasma of mice irradiated with 0.1 and 2 Gy. Several of these pathways have already been associated with bystander responses. CONCLUSION: Low and high dose effects overlapped both in EV-mediated alterations in signaling pathways leading to RIBE and in their systemic manifestations.