Postmortem chondrocyte viability in porcine articular cartilage: Influence of time, temperature, and burial under winter conditions.

The aim of the present study was to investigate the effects of time, temperature, and burial in a natural environment on the viability of chondrocytes in porcine femoral condyles using confocal laser scanning microscopy. Hind trotters from 10 pigs were buried or left unburied. Samples were collected daily and stained with a combination of vital dyes (calcein-AM and ethidium homodimer-1). The chondrocytes showed an intense staining corresponding to their vitality. In the first 3 days, viability decreased slowly and showed no statistical difference between buried and unburied samples. After the first 3 days, it decreased rapidly, with the viability of the buried samples being 66% on day 4, decreasing to 25% on day 8 and to 16% on day 10, while in the unburied samples it decreased to 43% on day 4, 13% on day 8 and 5% on day 10. Our results indicate a time, temperature, and burial dependent decrease in chondrocyte viability and suggest the use of chondrocyte viability as a marker for estimating PMI in both the natural environment and in animals, as well as its potential use in humans.

The Role of c-Abl Tyrosine Kinase in Brain and Its Pathologies.

Differentiated status, low regenerative capacity and complex signaling make neuronal tissues highly susceptible to translating an imbalance in cell homeostasis into cell death. The high rate of neurodegenerative diseases in the elderly population confirms this. The multiple and divergent signaling cascades downstream of the various stress triggers challenge researchers to identify the central components of the stress-induced signaling pathways that cause neurodegeneration. Because of their critical role in cell homeostasis, kinases have emerged as one of the key regulators. Among kinases, non-receptor tyrosine kinase (Abelson kinase) c-Abl appears to be involved in both the normal development of neural tissue and the development of neurodegenerative pathologies when abnormally expressed or activated. However, exactly how c-Abl mediates the progression of neurodegeneration remains largely unexplored. Here, we summarize recent findings on the involvement of c-Abl in normal and abnormal processes in nervous tissue, focusing on neurons, astrocytes and microglial cells, with particular reference to molecular events at the interface between stress signaling, DNA damage, and metabolic regulation. Because inhibition of c-Abl has neuroprotective effects and can prevent neuronal death, we believe that an integrated view of c-Abl signaling in neurodegeneration could lead to significantly improved treatment of the disease.

Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS.

Nuclear to cytoplasmic mislocalization and aggregation of multiple RNA-binding proteins (RBPs), including FUS, are the main neuropathological features of the majority of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobular degeneration (FTLD). In ALS-FUS, these aggregates arise from disease-associated mutations in FUS, whereas in FTLD-FUS, the cytoplasmic inclusions do not contain mutant FUS, suggesting different molecular mechanisms of FUS pathogenesis in FTLD that remain to be investigated. We have previously shown that phosphorylation of the C-terminal Tyr526 of FUS results in increased cytoplasmic retention of FUS due to impaired binding to the nuclear import receptor TNPO1. Inspired by the above notions, in the current study we developed a novel antibody against the C-terminally phosphorylated Tyr526 FUS (FUSp-Y526) that is specifically capable of recognizing phosphorylated cytoplasmic FUS, which is poorly recognized by other commercially available FUS antibodies. Using this FUSp-Y526 antibody, we demonstrated a FUS phosphorylation-specific effect on the cytoplasmic distribution of soluble and insoluble FUSp-Y526 in various cells and confirmed the involvement of the Src kinase family in Tyr526 FUS phosphorylation. In addition, we found that FUSp-Y526 expression pattern correlates with active pSrc/pAbl kinases in specific brain regions of mice, indicating preferential involvement of cAbl in the cytoplasmic mislocalization of FUSp-Y526 in cortical neurons. Finally, the pattern of immunoreactivity of active cAbl kinase and FUSp-Y526 revealed altered cytoplasmic distribution of FUSp-Y526 in cortical neurons of post-mortem frontal cortex tissue from FTLD patients compared with controls. The overlap of FUSp-Y526 and FUS signals was found preferentially in small diffuse inclusions and was absent in mature aggregates, suggesting possible involvement of FUSp-Y526 in the formation of early toxic FUS aggregates in the cytoplasm that are largely undetected by commercially available FUS antibodies. Given the overlapping patterns of cAbl activity and FUSp-Y526 distribution in cortical neurons, and cAbl induced sequestration of FUSp-Y526 into G3BP1 positive granules in stressed cells, we propose that cAbl kinase is actively involved in mediating cytoplasmic mislocalization and promoting toxic aggregation of wild-type FUS in the brains of FTLD patients, as a novel putative underlying mechanism of FTLD-FUS pathophysiology and progression.

Interactome screening of C9orf72 dipeptide repeats reveals VCP sequestration and functional impairment by polyGA.

Repeat expansions in the C9orf72 gene are a common cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, two devastating neurodegenerative disorders. One of the proposed mechanisms of GGGGCC repeat expansion is their translation into non-canonical dipeptide repeats, which can then accumulate as aggregates and contribute to these pathologies. There are five different dipeptide repeat proteins (polyGA, polyGR, polyPR, polyPA and polyGP), some of which are known to be neurotoxic. In the present study, we used BioID2 proximity labelling to identify the interactomes of all five dipeptide repeat proteins consisting of 125 repeats each. We identified 113 interacting partners for polyGR, 90 for polyGA, 106 for polyPR, 25 for polyPA and 27 for polyGP. Gene Ontology enrichment analysis of the proteomic data revealed that these target interaction partners are involved in a variety of functions, including protein translation, signal transduction pathways, protein catabolic processes, amide metabolic processes and RNA-binding. Using autopsy brain tissue from patients with C9orf72 expansion complemented with cell culture analysis, we evaluated the interactions between polyGA and valosin containing protein (VCP). Functional analysis of this interaction revealed sequestration of VCP with polyGA aggregates, altering levels of soluble valosin-containing protein. VCP also functions in autophagy processes, and consistent with this, we observed altered autophagy in cells expressing polyGA. We also observed altered co-localization of polyGA aggregates and p62 in cells depleted of VCP. All together, these data suggest that sequestration of VCP with polyGA aggregates contributes to the loss of VCP function, and consequently to alterations in autophagy processes in C9orf72 expansion disorders.

Intracellular Responses Triggered by Cold Atmospheric Plasma and Plasma-Activated Media in Cancer Cells.

Cold atmospheric plasma (CAP), an ionized gas operating at room temperature, has been increasingly studied with respect to its potential use in medicine, where its beneficial effects on tumor reduction in oncology have been demonstrated. This review discusses the cellular changes appearing in cell membranes, cytoplasm, various organelles, and DNA content upon cells' direct or indirect exposure to CAP or CAP-activated media/solutions (PAM), respectively. In addition, the CAP/PAM impact on the main cellular processes of proliferation, migration, protein degradation and various forms of cell death is addressed, especially in light of CAP use in the oncology field of plasma medicine.

Cold atmospheric plasma induces stress granule formation via an eIF2α-dependent pathway.

Cold atmospheric plasma is an ionized gas that shows promise in regenerative medical treatments, yet the mechanisms underlying its effects are still poorly understood. Plasma treatment promotes cell growth or cell death depending on the cell type and exposure parameters. To date, no early cell response to plasma, such as stress granule (SG) formation has been addressed. Cytoplasmic SGs are formed as an immediate cell response to acute stress stimuli by recruitment of over 140 proteins intertwined with cytoplasmic RNAs that leads to transient suspension of protein translation. Encouraged by the plasma effects in regenerative medicine and oncology, the atmospheric pressure plasma jet with argon gas flow is being utilized to treat SH-SY5Y cells with an inducible expression of the stress granule marker G3BP1, to gain an insight into early cell response to plasma and SG formation dynamics. Plasma effectively induces SG formation in the exposed cells in a flow/time-dependent manner, with the SG assembly clearly prompted by plasma-induced oxidative stress. Plasma causes SG formation via eIF2α-signaling, which is repressed with the SG formation inhibitor ISRIB. This insight into the early cell response to plasma treatment may lead to improved therapies in regenerative medicine and cancer treatment.

The Impact of ALS-Associated Genes hnRNPA1, MATR3, VCP and UBQLN2 on the Severity of TDP-43 Aggregation.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder, characterized by cytoplasmic inclusions of RNA-binding protein TDP-43. Despite decades of research and identification of more than 50 genes associated with amyotrophic lateral sclerosis (ALS), the cause of TDP-43 translocation from the nucleus and its aggregation in the cytoplasm still remains unknown. Our study addressed the impact of selected ALS-associated genes on TDP-43 aggregation behavior in wild-type and aggregation prone TDP-43 in vitro cell models. These were developed by deleting TDP-43 nuclear localization signal and stepwise shortening its low-complexity region. The SH-SY5Y cells were co-transfected with the constructs of aggregation-prone TDP-43 and wild-type or mutant ALS-associated genes hnRNPA1, MATR3, VCP or UBQLN2. The investigated genes displayed a unique impact on TDP-43 aggregation, generating distinct types of cytoplasmic inclusions, similar to those already described as resembling prion strains, which could represent the basis for neurodegenerative disease heterogeneity.

A Bifunctional Molecule with Lectin and Protease Inhibitor Activities Isolated from Crataeva tapia Bark Significantly Affects Cocultures of Mesenchymal Stem Cells and Glioblastoma Cells.

Currently available drugs for treatment of glioblastoma, the most aggressive brain tumor, remain inefficient, thus a plethora of natural compounds have already been shown to have antimalignant effects. However, these have not been tested for their impact on tumor cells in their microenvironment-simulated cell models, e.g., mesenchymal stem cells in coculture with glioblastoma cell U87 (GB). Mesenchymal stem cells (MSC) chemotactically infiltrate the glioblastoma microenvironment. Our previous studies have shown that bone-marrow derived MSCs impair U87 growth and invasion via paracrine and cell-cell contact-mediated cross-talk. Here, we report on a plant-derived protein, obtained from Crataeva tapia tree Bark Lectin (CrataBL), having protease inhibitory/lectin activities, and demonstrate its effects on glioblastoma cells U87 alone and their cocultures with MSCs. CrataBL inhibited U87 cell invasion and adhesion. Using a simplified model of the stromal microenvironment, i.e., GB/MSC direct cocultures, we demonstrated that CrataBL, when added in increased concentrations, caused cell cycle arrest and decreased cocultured cells' viability and proliferation, but not invasion. The cocultured cells' phenotypes were affected by CrataBL via a variety of secreted immunomodulatory cytokines, i.e., G-CSF, GM-CSF, IL-6, IL-8, and VEGF. We hypothesize that CrataBL plays a role by boosting the modulatory effects of MSCs on these glioblastoma cell lines and thus the effects of this and other natural lectins and/or inhibitors would certainly be different in the tumor microenvironment compared to tumor cells alone. We have provided clear evidence that it makes much more sense testing these potential therapeutic adjuvants in cocultures, mimicking heterogeneous tumor-stroma interactions with cancer cells in vivo. As such, CrataBL is suggested as a new candidate to approach adjuvant treatment of this deadly tumor.

Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies.

The GGGGCC (G4C2) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G4C2) n repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G4C2) n RNA foci. Overexpression of (G4C2)72 RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G4C2)72 RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1 Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G4C2) n RNA foci. In conclusion, (G4C2) n RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation.

Could a plant derived protein potentiate the anticancer effects of a stem cell in brain cancer?

Glioblastoma is the most aggressive brain tumor with poor overall survival bellow 2 years. The natural compounds with anti-cancer properties, are thus gaining attention for possible adjuvant GBM treatment. In various cancer models Enterolobium contortisiliquum Trypsin Inhibitor (EcTI) proved to have anti-cancer effects. Here, we investigated the EcTI effects on GBM U87 cells and on mesenchymal stem cells (MSC) compared to their direct coculture (MSC/U87). MSC are present in tumor stroma, modulating GBM cells phenotype, and also represent potential drug delivery vehicle due to their tumor tropism. We showed that in p53-wild type U87 cells, metabolic activity was less affected by EcTI as in MSC monocuture, but the metabolic rate of mixed coculture was significantly reduced at lower EcTI concentration. Under coculture condition, EcTI potentiated MSC induced cell cycle arrest, possible due to highly increased p53, p21 and lower D1 expression, but there was no effect on apoptosis. Accordingly, in the coculture EcTI also enhanced Ca2+ signalling mediated via bradykinin receptor 2, being associated with nitric oxide release that highly impaired proliferation and invasion. The mechanism did not seem to involve changes in cell adhesion but rather it down-regulated the ß1 integrin signaling with associated p-FAK in U87 cells, both supporting inhibition of invasion. Finally, some cytokines were down-regulated, indicating that EcTI inhibition of signalling might be mediated by cytokines. In conclusion, these results indicate that in cocultured MSC/U87 cells EcTI impairs the metabolic activity, proliferation, and reduced invasion, possibly associated with observed cytokines secretion. In this context, we confirmed that the plant derived protein potentiated the anticancer effects, induced by MSC, as represented by GBM U87 cell line.

Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model.

Familial dysautonomia (FD) is a rare genetic disease with no treatment, caused by an intronic point mutation (c.2204+6T>C) that negatively affects the definition of exon 20 in the elongator complex protein 1 gene (ELP1 also known as IKBKAP). This substitution modifies the 5' splice site and, in combination with regulatory splicing factors, induces different levels of exon 20 skipping, in various tissues. Here, we evaluated the therapeutic potential of a novel class of U1 snRNA molecules, exon-specific U1s (ExSpeU1s), in correcting ELP1 exon 20 recognition. Lentivirus-mediated expression of ELP1-ExSpeU1 in FD fibroblasts improved ELP1 splicing and protein levels. We next focused on a transgenic mouse model that recapitulates the same tissue-specific mis-splicing seen in FD patients. Intraperitoneal delivery of ELP1-ExSpeU1s-adeno-associated virus particles successfully increased the production of full-length human ELP1 transcript and protein. This splice-switching class of molecules is the first to specifically correct the ELP1 exon 20 splicing defect. Our data provide proof of principle of ExSpeU1s-adeno-associated virus particles as a novel therapeutic strategy for FD.

Kinin-B1 Receptor Stimulation Promotes Invasion and is Involved in Cell-Cell Interaction of Co-Cultured Glioblastoma and Mesenchymal Stem Cells.

Glioblastoma multiforme (GBM) represents the most lethal brain tumour, and these tumours have very limited treatment options. Mesenchymal stem cells (MSC) are considered as candidates for advanced cell therapies, due to their tropism towards GBM, possibly affecting their malignancy, thus also representing a potential therapeutic vector. Therefore, we aimed to compare the effects of bone-marrow-derived versus adipose-tissue-derived MSC (BM-/AT-MSC) on heterogeneous populations of tumour cells. This cells' interplay was addressed by the in-vitro two-dimensional (monolayer) and three-dimensional (spheroid) co-culture models, using U87 and U373 GBM cell lines, expressing genotypically different mesenchymal transcriptome profiles. U87 cell low mesenchymal profile expressed high levels of kinin receptor 1 (B1R) and their invasion was greatly enhanced by the B1R agonist des-Arg9-bradykinin upon BM-MSC co-culturing in 3D co-cultures. This correlated to significantly higher cell-cell interactions in U87/BM-MSC mixed spheroids. This was not observed with the U373 cells and not in AT-MSC co-cultures. Altogether, these data support the on-going exploration of B1R as target for adjuvant approach in GBM therapy. Secondly, the results emphasize the need for further careful exploration of the selectivity regarding the origin of MSC as potential candidates for cell therapies, particular in cancer, where they may adversely affect heterogeneous tumour cell populations.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7.

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.

Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell lines.

Glioblastoma multiforme are an aggressive form of brain tumors that are characterized by distinct invasion of single glioblastoma cells, which infiltrate the brain parenchyma. This appears to be stimulated by the communication between cancer and stromal cells. Mesenchymal stem cells (MSCs) are part of the glioblastoma microenvironment, and their 'cross-talk' with glioblastoma cells is still poorly understood. Here, we examined the effects of bone marrow-derived MSCs on two different established glioblastoma cell lines U87 and U373. We focused on mutual effects of direct MSC/glioblastoma contact on cellular invasion in three-dimensional invasion assays in vitro and in a zebrafish embryo model in vivo. This is the first demonstration of glioblastoma cell-type-specific responses to MSCs in direct glioblastoma co-cultures, where MSCs inhibited the invasion of U87 cells and enhanced the invasion of U373. Inversely, direct cross-talk between MSCs and both of glioblastoma cell lines enhanced MSC motility. MSC-enhanced invasion of U373 cells was assisted by overexpression of proteases cathepsin B, calpain1, uPA/uPAR, MMP-2, -9 and -14, and increased activities of some of these proteases, as determined by the effects of their selective inhibitors on invasion. In contrast, these proteases had no effect on U87 cell invasion under MSC co-culturing. Finally, we identified differentially expressed genes, in U87 and U373 cells that could explain different response of these cell lines to MSCs. In conclusion, we demonstrated that MSC/glioblastoma cross-talk is different in the two glioblastoma cell phenotypes, which contributes to tumor heterogeneity.

Cell Proliferation on Polyethylene Terephthalate Treated in Plasma Created in SO2/O2 Mixtures.

Samples of polymer polyethylene terephthalate were exposed to a weakly ionized gaseous plasma to modify the polymer surface properties for better cell cultivation. The gases used for treatment were sulfur dioxide and oxygen of various partial pressures. Plasma was created by an electrodeless radio frequency discharge at a total pressure of 60 Pa. X-ray photoelectron spectroscopy showed weak functionalization of the samples' surfaces with the sulfur, with a concentration around 2.5 at %, whereas the oxygen concentration remained at the level of untreated samples, except when the gas mixture with oxygen concentration above 90% was used. Atomic force microscopy revealed highly altered morphology of plasma-treated samples; however, at high oxygen partial pressures this morphology vanished. The samples were then incubated with human umbilical vein endothelial cells. Biological tests to determine endothelialization and possible toxicity of the plasma-treated polyethylene terephthalate samples were performed. Cell metabolic activity (MTT) and in vitro toxic effects of unknown compounds (TOX) were assayed to determine the biocompatibility of the treated substrates. The biocompatibility demonstrated a well-pronounced maximum versus gas composition which correlated well with development of the surface morphology.

Proteases and cytokines as mediators of interactions between cancer and stromal cells in tumours.

Proteolytic enzymes are highly relevant in different processes of cancer progression. Their interplay with other signalling molecules such as cytokines represents important regulation of multicellular cross-talk. In this review, we discuss protease regulation mechanisms of cytokine signalling in various types of cancer. Additionally, we highlight the reverse whereby cytokines have an impact on protease expression in an autocrine and paracrine manner, representing complex feedback mechanisms among multiple members of these two protein families. The relevance of the protease-cytokine axis is illustrated in glioblastoma, where interactions between normal mesenchymal stem cells and cancer cells play an important role in this very malignant form of brain cancer.

Improved Protective Effect of Umbilical Cord Stem Cell Transplantation on Cisplatin-Induced Kidney Injury in Mice Pretreated with Antithymocyte Globulin.

Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone.

Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells.

The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.

Glioblastoma-mesenchymal stem cell communication modulates expression patterns of kinin receptors: Possible involvement of bradykinin in information flow.

The most aggressive subtype of brain tumors is glioma WHO grade IV, the glioblastoma (GBM). The present work aims to elucidate the role of kinin receptors in interactions between GBM cells and mesenchymal stem cells (MSC). The GBM cell line U87-MG was stably transfected to express dsRed protein, single cell cloned, expanded, and cultured with MSC, both in the direct co-cultures (DC) and indirect co-cultures (IC) at equal cell number ratio for 72 h. Up- and down-regulation of matrix metalloproteases (MMP)-9 expression in U87-MG and MSC cells, respectively, in direct co-culture points to possible MSC participation in tumor invasion. MMP9 expression is in line with significantly increased expression of kinin B1 (B1R) and B2 receptor (B2R) in U87-MG cells and their decreased levels in MSC, as confirmed by quantitative assessment using flow cytometric analysis. Similarly, in indirect cultures (IC), lacking the contact between GBM and MSC cells, an increase of B1 and B2 receptor expression was again noted in U87-MG cells, and no significant changes in kinin receptors in MSC was observed. Functionality of kinin-B1 and B2 receptors was evidenced by stimulation of intracellular calcium fluxes by their respective agonists, des-Arg9-bradykinin (DBK) and bradykinin (BK). Moreover, BK showed a feedback control on kinin receptor expression in mono-cultures, direct and indirect co-cultures. The treatment with BK resulted in down-regulation of B1 and B2 receptors in MSC, with simultaneous up-regulation of these receptors in U87-MG cells, suggesting that functions of BK in information flow between these cells is important for tumor progression and invasion. © 2015 International Society for Advancement of Cytometry.