New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Effect of radiotherapy on the DNA cargo and cellular uptake mechanisms of extracellular vesicles.

In the past decades, plenty of evidence has gathered pointing to the role of extracellular vesicles (EVs) secreted by irradiated cells in the development of radiation-induced non-targeted effects. EVs are complex natural structures composed of a phospholipid bilayer which are secreted by virtually all cells and carry bioactive molecules. They can travel certain distances in the body before being taken up by recipient cells. In this review we discuss the role and fate of EVs in tumor cells and highlight the importance of DNA specimens in EVs cargo in the context of radiotherapy. The effect of EVs depends on their cargo, which reflects physiological and pathological conditions of donor cell types, but also depends on the mode of EV uptake and mechanisms involved in the route of EV internalization. While the secretion and cargo of EVs from irradiated cells has been extensively studied in recent years, their uptake is much less understood. In this review, we will focus on recent knowledge regarding the EV uptake of cancer cells and the effect of radiation in this process.

The miRNA Content of Bone Marrow-Derived Extracellular Vesicles Contributes to Protein Pathway Alterations Involved in Ionising Radiation-Induced Bystander Responses.

Extracellular vesicles (EVs), through their cargo, are important mediators of bystander responses in the irradiated bone marrow (BM). MiRNAs carried by EVs can potentially alter cellular pathways in EV-recipient cells by regulating their protein content. Using the CBA/Ca mouse model, we characterised the miRNA content of BM-derived EVs from mice irradiated with 0.1 Gy or 3 Gy using an nCounter analysis system. We also analysed proteomic changes in BM cells either directly irradiated or treated with EVs derived from the BM of irradiated mice. Our aim was to identify key cellular processes in the EV-acceptor cells regulated by miRNAs. The irradiation of BM cells with 0.1 Gy led to protein alterations involved in oxidative stress and immune and inflammatory processes. Oxidative stress-related pathways were also present in BM cells treated with EVs isolated from 0.1 Gy-irradiated mice, indicating the propagation of oxidative stress in a bystander manner. The irradiation of BM cells with 3 Gy led to protein pathway alterations involved in the DNA damage response, metabolism, cell death and immune and inflammatory processes. The majority of these pathways were also altered in BM cells treated with EVs from mice irradiated with 3 Gy. Certain pathways (cell cycle, acute and chronic myeloid leukaemia) regulated by miRNAs differentially expressed in EVs isolated from mice irradiated with 3 Gy overlapped with protein pathway alterations in BM cells treated with 3 Gy EVs. Six miRNAs were involved in these common pathways interacting with 11 proteins, suggesting the involvement of miRNAs in the EV-mediated bystander processes. In conclusion, we characterised proteomic changes in directly irradiated and EV-treated BM cells, identified processes transmitted in a bystander manner and suggested miRNA and protein candidates potentially involved in the regulation of these bystander processes.

RENEB Inter-Laboratory Comparison 2021: The Gamma-H2AX Foci Assay.

The Running the European Network of biological and retrospective dosimetry (RENEB) network of laboratories has a range of biological and physical dosimetry assays that can be deployed in the event of a radiation incident to provide exposure assessment. To maintain operational capability and provide training, RENEB runs regular inter-laboratory comparison (ILC) exercises. The RENEB ILC2021 was carried out with all the biological and physical dosimetry assays employed in the network. The focus of this paper is to evaluate the results from 6 laboratories that took part using the gamma-H2AX radiation-induced foci assay. For two laboratories this was their first RENEB ILC. Blood samples were homogenously exposed to 240 kVp X rays (1 Gy/min) to provide calibration data, (0-4 Gy), and a few weeks later three blind coded test samples, (0, 1.2 and 3.5 Gy) were prepared. All samples were allowed a 2 h repair time at 37°C before being transported, on ice packs, to the participating laboratories. On arrival, the samples were processed, scored either manually or automatically for gamma-H2AX foci and dose estimates for the 3 blind coded samples sent to the organizing laboratory. The temperature of samples during transit and the time taken to report the dose estimates were recorded. Subsequent examination of the data from each laboratory used the doses estimates to assign triage categories to the samples. After receipt of the samples, the quickest report of dose estimates was 4.6 h. Analysis of variance revealed that the laboratory carrying out the assay had a significant effect on the foci yield (P < 0.001) for the calibration data, but not on the dose estimates of the blind coded samples (P = 0.101). All laboratories correctly identified the unirradiated and irradiated samples, although the dose estimates for the latter tended to under-estimate the dose. Two participants seriously under-estimated the dose for the highly exposed sample, which resulted in the sample being placed in the lowest triage category not the highest. However, this under-estimation resulted from the samples not remaining cold during shipment, due to a delay in transit and was not related to the experience of the participating laboratory. Overall, the RENEB network laboratories have demonstrated it is possible to quickly identify a recent whole-body acute exposure using the gamma-H2AX assay within the conditions of the ILC. In addition, an ILC provides a useful training and harmonization exercise for laboratories.

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.

Nuclear Syndecan-1 Regulates Epithelial-Mesenchymal Plasticity in Tumor Cells.

Tumor cells undergoing epithelial-mesenchymal transition (EMT) lose cell surface adhesion molecules and gain invasive and metastatic properties. EMT is a plastic process and tumor cells may shift between different epithelial-mesenchymal states during metastasis. However, how this is regulated is not fully understood. Syndecan-1 (SDC1) is the major cell surface proteoglycan in epithelial cells and has been shown to regulate carcinoma progression and EMT. Recently, it was discovered that SDC1 translocates into the cell nucleus in certain tumor cells. Nuclear SDC1 inhibits cell proliferation, but whether nuclear SDC1 contributes to the regulation of EMT is not clear. Here, we report that loss of nuclear SDC1 is associated with cellular elongation and an E-cadherin-to-N-cadherin switch during TGF-ß1-induced EMT in human A549 lung adenocarcinoma cells. Further studies showed that nuclear translocation of SDC1 contributed to the repression of mesenchymal and invasive properties of human B6FS fibrosarcoma cells. The results demonstrate that nuclear translocation contributes to the capacity of SDC1 to regulate epithelial-mesenchymal plasticity in human tumor cells and opens up to mechanistic studies to elucidate the mechanisms involved.

Syndecan-1 Overexpressing Mesothelioma Cells Inhibit Proliferation, Wound Healing, and Tube Formation of Endothelial Cells.

Malignant mesothelioma (MM) is an aggressive tumor of the serosal cavities. Angiogenesis is important for mesothelioma progression, but so far, anti-angiogenic agents have not improved patient survival. Our hypothesis is that better understanding of the regulation of angiogenesis in this tumor would largely improve the success of such a therapy. Syndecan-1 (SDC-1) is a transmembrane heparan sulfate proteoglycan that acts as a co-receptor in various cellular processes including angiogenesis. In MM, the expression of SDC-1 is generally low but when present, SDC-1 associates to epithelioid differentiation, inhibition of tumor cell migration and favorable prognosis, meanwhile SDC-1 decrease deteriorates the prognosis. In the present study, we studied the effect of SDC-1 overexpression and silencing on MM cells ability to secrete angiogenic factors and monitored the downstream effect of SDC-1 modulation on endothelial cells proliferation, wound healing, and tube formation. This was done by adding conditioned medium from SDC-1 transfected and SDC-1 silenced mesothelioma cells to endothelial cells. Moreover, we investigated the interplay and molecular functional changes in angiogenesis in a co-culture system and characterized the soluble angiogenesis-related factors secreted to the conditioned media. We demonstrated that SDC-1 over-expression inhibited the proliferation, wound healing, and tube formation of endothelial cells. This effect was mediated by a multitude of angiogenic factors comprising angiopoietin-1 (Fold change ± SD: 0.65 ± 0.07), FGF-4 (1.45 ± 0.04), HGF (1.33 ± 0.07), NRG1-ß1 (1.35 ± 0.08), TSP-1 (0.8 ± 0.02), TIMP-1 (0.89 ± 0.01) and TGF-ß1 (1.35 ± 0.01). SDC-1 silencing increased IL8 (1.33 ± 0.06), promoted wound closure, but did not influence the tube formation of endothelial cells. Pleural effusions from mesothelioma patients showed that Vascular Endothelial Growth Factor (VEGF) levels correlate to soluble SDC-1 levels and have prognostic value. In conclusion, SDC-1 over-expression affects the angiogenic factor secretion of mesothelioma cells and thereby inhibits endothelial cells proliferation, tube formation, and wound healing. VEGF could be used in prognostic evaluation of mesothelioma patients together with SDC-1.

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.

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.

Mitochondrial DNA damage in the hair bulb: can it be used as a noninvasive biomarker of local exposure to low LET ionizing radiation?

Purpose: Our aim was to evaluate whether mitochondrial DNA (mtDNA) damage in hair bulbs could be a suitable biomarker for the detection of local exposure to ionizing radiation.Materials and methods: Mouse hair was collected 4 and 24 hours, 3 and 10 days after single whole-body exposure to 0, 0.1, and 2 Gy radiation. Pubic hair (treated area) and scalp hair (control area) were collected from 13 prostate cancer patients before and after fractioned radiotherapy with an average total dose of 2.7 Gy to follicles after five fractions. Unspecified lesion frequency of mtDNA was analyzed with long PCR, large mtDNA deletion levels were tested with real-time PCR.Results: Unspecified lesion frequency of mtDNA significantly increased in mouse hair 24 hours after irradiation with 2 Gy, but variance among samples was high. No increase in lesion frequency could be detected after 0.1 Gy irradiation. In prostate cancer patients, there was no significant change in either the unspecified lesion frequency or in the proportion of 4934-bp deleted mtDNA in pubic hair after radiotherapy. The proportions of murine 3860-bp common deletion, human 4977-bp common deletion and 7455-bp deleted mtDNA were too low to be analyzed reliably.Conclusions: Our results suggest that the unspecified lesion frequency and proportion of large deletions of mtDNA in hair bulbs are not suitable biomarkers of exposure to ionizing radiation.

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.

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.

In Vivo Irradiation of Mice Induces Activation of Dendritic Cells.

It is becoming clear that ionizing radiation positively influences certain immune parameters, which opens the possibility for combining radio- and immunotherapies in cancer treatment. The presence of functionally competent dendritic cells (DCs) is crucial in mounting a successful antitumor immune response. While it has been shown that DCs are relatively radioresistant, few and contradictory data are available on how ionizing radiation alters the functional integrity of these cells. Therefore, our objective was to investigate the effect of whole-body irradiation on the function of splenic DCs. C57Bl/6 mice were irradiated with 0.1, 0.25, and 2 Gy X-rays and changes in the phenotype of splenic DCs were compared to unirradiated controls. An increase was seen in DC surface markers influencing DC-T cell interactions. In vivo cytokine production was determined by direct intracellular cytokine staining. Irradiation with 2 Gy induced a 1.6-fold increase in IL-1α production, while the combination of irradiation and lipopolysaccharide (LPS) treatment induced a 3.9-fold increase, indicating a strong synergism between irradiation and LPS stimulation. Interaction of DCs with effector and regulatory T cells was investigated in a mixed lymphocyte reaction. While DCs from control animals induced stronger proliferation of regulatory T cells, DCs from animals irradiated with 2 Gy induced stronger proliferation of effector T cells. Antigen uptake and presentation was investigated by measuring the capacity of DCs to internalize and present ovalbumine (OVA)-derived peptides on their major histocompatibility complex (MHCI) molecules. Irradiation with 2 Gy did not influence antigen uptake or presentation, while low doses stimulated antigen uptake and reduced the level of antigen presentation. In conclusion, high-dose in vivo irradiation induced increased expression of T cell costimulatory markers, enhanced production of proinflammatory cytokines and a stronger stimulation of effector T cell proliferation than that of regulatory T cells. However, it did not influence DC antigen uptake or presentation. On the other hand, low-dose irradiation increased antigen uptake and lowered antigen presentation of DCs, indicating that low- and high-dose irradiation act on different pathways in DCs.

Molecular targets and signaling pathways regulated by nuclear translocation of syndecan-1.

BACKGROUND: The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. RESULTS: We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-ß pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκß, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix organization and transmembrane transport. CONCLUSION: Collectively, abrogation of nuclear translocation of syndecan-1 resulted in a set of changes clustering in distinct patterns, which highlighted the functional importance of nuclear syndecan-1 in hampering cell proliferation and the cell cycle. This study emphasizes the importance of the localization of syndecan-1 when considering its effects on tumor cell fate.

Ex vivo evaluation of tumor cell specific drug responses in malignant pleural effusions.

The effect of chemotherapy may be improved by combining the most effective drugs based on testing the sensitivity of the individual tumor ex vivo. Such estimations of tumor cells from effusions have so far not been implemented in the clinical routine as a basis for individualized choice of therapy. One obstacle for such analyses is the admixture of benign cells that might obscure the results. In this paper we test and compare two ways of performing the analysis specifically on tumor cells. First we enrich the tumor cells, using antibody labeled magnetic separation, and measure the effects of subsequent drug exposure with the metabolic activity assays WST-1 and alamar blue. The second way of estimating drug effects specifically on tumor cells employs multi parameter flow cytometry, measuring apoptosis with the propidium iodide / AnnexinV technique and, particularly for pemetrexed, possible effects on cell cycle progression in immunologically identified tumor cells. The two techniques produce similar results, indicating a possible use in personalized medicine. The possible predictive role of the analysis remains to be shown.

Ionizing Radiation-Induced Immune and Inflammatory Reactions in the Brain.

Radiation-induced late brain injury consisting of vascular abnormalities, demyelination, white matter necrosis, and cognitive impairment has been described in patients subjected to cranial radiotherapy for brain tumors. Accumulating evidence suggests that various degrees of cognitive deficit can develop after much lower doses of ionizing radiation, as well. The pathophysiological mechanisms underlying these alterations are not elucidated so far. A permanent deficit in neurogenesis, chronic microvascular alterations, and blood-brain barrier dysfunctionality are considered among the main causative factors. Chronic neuroinflammation and altered immune reactions in the brain, which are inherent complications of brain irradiation, have also been directly implicated in the development of cognitive decline after radiation. This review aims to give a comprehensive overview on radiation-induced immune alterations and inflammatory reactions in the brain and summarizes how these processes can influence cognitive performance. The available data on the risk of low-dose radiation exposure in the development of cognitive impairment and the underlying mechanisms are also discussed.

Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen.

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.

Chemosensitivity and resistance testing in malignant effusions with focus on primary malignant mesothelioma and metastatic adenocarcinoma.

Cell based chemosensitivity and resistance testing is an attractive approach that offers functional measurement of drug response ex vivo with the ultimate goal to guide the choice of chemotherapy for various cancers. Thus, it has a great potential to select patients for the optimal treatment option, thereby offering a tool for personalized cancer therapy. Despite several decades of intensive scientific efforts ex-vivo tests are still not incorporated in the standard of care. Limited access to fresh tumor tissue, unsatisfactory models and single readout as endpoint constitute major hindrance. Thus, establishing and validating clinically useful and reliable model systems still remains a major challenge. Here we present malignant effusions as valuable sources for ex-vivo chemosensitivity and resistance testing. Accumulation of a malignant effusion in the pleura, peritoneum or pericardium is often the first diagnostic material for both primary malignant mesothelioma and a broad spectrum of metastatic adenocarcinoma originating from lung-, breast-, ovary- and gastro-intestinal organs as well as lymphoma. In contrast to biopsies, in these effusions malignant cells are easily accessible and often abundant. Effusion derived cells can occur dissociated or forming three-dimensional papillary structures that authentically recapitulate the biology of the corresponding tumor tissue and offer models for ex vivo testing. In addition, effusions have the advantage of being available prior to or concurrent with the pathological review, thus constituting an excellent source of viable cells for simultaneous molecular profiling, biomarker analysis and for establishing primary cells for studying tumor biology and resistance mechanisms. For a reliable test, however, a careful validation is needed, taking into account the inherited heterogeneity of malignant tumors, but also the complex interplay between malignant and benign cells, which are always present in this setting.

Syndecan-1 in Cancer: Implications for Cell Signaling, Differentiation, and Prognostication.

Syndecan-1, a cell surface heparan sulfate proteoglycan, is critically involved in the differentiation and prognosis of various tumors. In this review, we highlight the synthesis, cellular interactions, and the signalling pathways regulated by syndecan-1. The basal syndecan-1 level is also crucial for understanding the sequential changes involving malignant transformation, tumor progression, and advanced or disseminated cancer stages. Moreover, we focus on the cellular localization of this proteoglycan as cell membrane anchored and/or shed, soluble syndecan-1 with stromal or nuclear accumulation and how this may carry different, highly tissue specific prognostic information for individual tumor types.

Syndecan-1 alters heparan sulfate composition and signaling pathways in malignant mesothelioma.

Syndecan-1 is a proteoglycan that acts as co-receptor through its heparan sulfate (HS) chains and plays important roles in cancer. HS chains are highly variable in length and sulfation pattern. This variability is enhanced by the SULF1/2 enzymes, which remove 6-O-sulfates from HS. We used malignant mesothelioma, an aggressive tumor with poor prognosis, as a model and demonstrated that syndecan-1 over-expression down-regulates SULF1 and alters the HS biosynthetic machinery. Biochemical characterization revealed a 2.7-fold reduction in HS content upon syndecan-1 over-expression, but an overall increase in sulfation. Consistent with low SULF1 levels, trisulfated disaccharides increased 2.5-fold. ERK1/2 activity was enhanced 6-fold. Counteracting ERK activation, Akt, WNK1, and c-Jun were inhibited. The net effect of these changes manifested in G1 cell cycle arrest. Studies of pleural effusions showed that SULF1 levels are lower in pleural malignancies compared to benign conditions and inversely correlate with the amounts of syndecan-1, suggesting important roles for syndecan-1 and SULF1 in malignant mesothelioma.