Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties.
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ABSTRACT:
SUBMITTER: McCoy MG
PROVIDER: S-EPMC6588602 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
Publications
Endothelial cells promote 3D invasion of GBM by IL-8-dependent induction of cancer stem cell properties.
McCoy Michael G MG Nyanyo Dennis D Hung Carol K CK Goerger Julian Palacios JP R Zipfel Warren W Williams Rebecca M RM Nishimura Nozomi N Fischbach Claudia C
Scientific reports 20190621 1
Rapid growth and perivascular invasion are hallmarks of glioblastoma (GBM) that have been attributed to the presence of cancer stem-like cells (CSCs) and their association with the perivascular niche. However, the mechanisms by which the perivascular niche regulates GBM invasion and CSCs remain poorly understood due in part to a lack of relevant model systems. To simulate perivascular niche conditions and analyze consequential changes of GBM growth and invasion, patient-derived GBM spheroids wer ...[more]
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Project description:Glioblastoma multiforme (GBM), the most advanced form of a large subset of brain tumors collectively known as glioma, is the most aggressive and invasive type of brain tumor. Patients usually experience a median survival range of 9 to 12 months. GBMs are extremely difficult to manage because of the tumor cells’ tendency to migrate and pervade into adjacent tissues. Surgical resection of GBMs generally only slows disease progression because any remaining tumor cells proceed to migrate through brain tissues and reform a new tumor mass. It is hypothesized that the invasive phenotype of these tumor cells may be attributable to unique gene expression. Stationary core and invasive rim tumor cells were collected separately by laser capture microdissection (LCM) from 19 biopsy samples. Identification of differentially expressed genes in the tumor core and invasive rim can give valuable insight to the genes and pathways potentially involved with the invasive phenotype. This information can then be used to generate possible biomarkers, diagnostic markers, or drug targets. Human glioblastoma tumor samples were obtained from patients who underwent primary therapeutic brain tumor resection. All specimens were collected and submitted to the study under institutional review board approved protocols. None of the patients had been subjected to chemotherapy or radiotherapy prior to resection, in order to avoid genetic signatures that are due to exposure to alkylating agents and/or ionizing radiation. All specimens were verified as GBMs by a neuropathologist. Tumors are embedded in OCT, then sectioned in a cryostat at -20C, to a thickness of 8-10 um and placed onto HistoGene slides. Sections to be microdissected are removed from the -80C fixed and stained with an abbreviated Hematoxylin and Eosin protocol. Two thousand tumor core and invasive cells are dissected onto separate caps using the PixCell II instrument using CapSure™ Macro LCM Caps. The CapSure™ Macro LCM Caps LCM 0211 should be used with the AutoPix instrument. Cells in the tumor core are identified by nuclear atypia and size and captured using the larger spot sizes. Tumor cells immediately adjacent to necrotic areas, cortical areas, cells with small regular nuclei, endothelial and blood cells should be avoided. Individual white matter invading GBM cells can be identified by means of their nuclear atypia and heteropyknotic staining, which is consistent with that of the cells within the tumor core. They should be microdissected using the 7.5 um laser spot size and the initial power settings recommended by the PixCell II manual. After microdissection all harvested material should immediately be lysed on the cap by applying the lysis buffer (XB) from the PicoPure RNA Isolation Kit according to the PicoPure protocol and stored -80C. Cell populations harvested on different caps can be pooled at the time of RNA isolation. RNA integrity is varified by identification of distinct 28S and 18S ribosomal bands with an Agilent Bioanalyzer using the RNA 6000 Nano LabChip kit. Total RNA was isolated from 2000 LCM cells (to ensure at least 500 ng total RNA) using the PicoPure RNA solation Kit, following manufacturers protocol. mRNA is reverse transcribed with the RiboAmp RNA Amplification kit. 500 ng total RNA is amplified with the RiboAmp RNA Amplification kit, following manufacturer’s instructions. The total yield that can be expected falls between 30 and 60 µg copy RNA. The size of the copy RNA should be verified by gel electrophoresis. Approximately 500ng of copy RNA can be separated on a 1% agarose gel in TAE buffer. A smear of amplified material should be seen between 200 and 3000bp. Six µg amplified RNA are labeled in a RT with SuperScriptIII in the presence of dUTP Cy5 utilizing 6 µg random hexamers as primers. Universal reference RNA is amplified one round in the same manner and labeled with Cy3 dUTP. Labeled cDNA is hybridized overnight onto cDNA microarray. Following hybridization, arrays are washed, scanned and quantitated with the Axon GenePix 4000 microarray reader (Axon Instruments). Gene expression results are analyzed using GeneSpring (Silicon Genetics) software. Intensity dependent normalization is applied, where the ratio is reduced to the residual Lowess fit of the intensity versus ratio curve. The measured intensity of each gene is divided by its reference channel (signal from the universal reference RNA) in each sample. When the reference channel is below 10, the data point is considered uninformative. The ratios (sample over reference) for the tumor core experiments and invasive rim experiments are averaged and compared. Genes that are more than two-fold differentially regulated in the majority of the matched core/invasive rim sets are selected.
| S-ECPF-GEOD-12689 | biostudies-other
Project description:Glioblastoma (GBM) is the most common malignant primary brain tumor in adults, which is fast growing and tends to invade surrounding normal brain tissues. Uncovering the molecular and cellular mechanisms of GBM high invasion potential is of great importance for the treatment and prognostic prediction. However, the commonly used two-dimensional (2D) cell culture and analysis system suffers from lack of the heterogeneity and in vivo property of brain tissues. Here, we established a three-dimensional (3D) cell culture-based analysis system that could better recapitulate the heterogeneity of GBM and mimic the in vivo conditions in the brain. The GBM cell lines, DBTRG and U251, were cultured by hanging drop culture into the GBM multicellular spheroids, which were embedded in the optimized 3D brain-stiffness-mimicking matrix gel (0.5 mg/ml Collagen Ⅰ + 3 mg/ml Matrigel+ 3.3 mg/ml Hyaluronic Acid (HA)). The biochemical composition of the optimized matrix gel is similar to that of the brain microenvironment, and the elastic modulus is close to that of the brain tissue. The dynamics of the GBM spheroids was examined using high-content imaging for 60 h, and four metrics including invasion distance, invasion area, single-cell invasion velocity, and directionality were employed to quantify the invasion capacity. The result showed that DBTRG cells possess higher invasion capacity than U251 cells, which was consistent with the results of the classic transwell test. Transcriptome analysis of both cell lines was performed to explore the underlying molecular mechanisms. Our novel brain-stiffness-mimicking matrix gel enables comprehensive invasion analysis of the 3D cultured GBM cells and provides a model basis for in-depth exploration of the mechanisms regulating GBM invasion including the interaction between GBM cells and brain stroma.
| S-EPMC9218788 | biostudies-literature
Project description:Zika virus (ZIKV)3 is an enveloped, single-stranded, positive-sense RNA virus of the Flaviviridae family that has emerged as a public health threat because of its global transmission and link to microcephaly. Currently there is no vaccine for this virus. Conversion of cholesterol to 25-hydroxycholesterol by cholesterol 25-hydroxylase (CH25H) has been shown to have broad antiviral properties. However, the molecular basis of induction of CH25H in humans is not known. Elucidation of signaling and transcriptional events for induction of CH25H expression is critical for designing therapeutic antiviral agents. In this study, we show that CH25H is induced by ZIKV infection or Toll-like receptor stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines, including IL-1β, tumor necrosis factor α, and IL-6, and this induction depends on the STAT1 transcription factor. Additionally, we observed that cAMP-dependent transcription factor (ATF3) weakly binds to the CH25H promoter, suggesting cooperation with STAT1. However, ZIKV-induced CH25H was independent of type I interferon. These findings provide important information for understanding how the Zika virus induces innate inflammatory responses and promotes the expression of anti-viral CH25H protein.
| S-EPMC6779448 | biostudies-literature
Project description:Glioblastoma multiforme (GBM), the most advanced form of a large subset of brain tumors collectively known as glioma, is the most aggressive and invasive type of brain tumor. Patients usually experience a median survival range of 9 to 12 months. GBMs are extremely difficult to manage because of the tumor cellsâ tendency to migrate and pervade into adjacent tissues. Surgical resection of GBMs generally only slows disease progression because any remaining tumor cells proceed to migrate through brain tissues and reform a new tumor mass. It is hypothesized that the invasive phenotype of these tumor cells may be attributable to unique gene expression. Stationary core and invasive rim tumor cells were collected separately by laser capture microdissection (LCM) from 19 biopsy samples. Identification of differentially expressed genes in the tumor core and invasive rim can give valuable insight to the genes and pathways potentially involved with the invasive phenotype. This information can then be used to generate possible biomarkers, diagnostic markers, or drug targets. Human glioblastoma tumor samples were obtained from patients who underwent primary therapeutic brain tumor resection. All specimens were collected and submitted to the study under institutional review board approved protocols. None of the patients had been subjected to chemotherapy or radiotherapy prior to resection, in order to avoid genetic signatures that are due to exposure to alkylating agents and/or ionizing radiation. All specimens were verified as GBMs by a neuropathologist. Tumors are embedded in OCT, then sectioned in a cryostat at -20C, to a thickness of 8-10 um and placed onto HistoGene slides. Sections to be microdissected are removed from the -80C fixed and stained with an abbreviated Hematoxylin and Eosin protocol. Two thousand tumor core and invasive cells are dissected onto separate caps using the PixCell II instrument using CapSure⢠Macro LCM Caps. The CapSure⢠Macro LCM Caps LCM 0211 should be used with the AutoPix instrument. Cells in the tumor core are identified by nuclear atypia and size and captured using the larger spot sizes. Tumor cells immediately adjacent to necrotic areas, cortical areas, cells with small regular nuclei, endothelial and blood cells should be avoided. Individual white matter invading GBM cells can be identified by means of their nuclear atypia and heteropyknotic staining, which is consistent with that of the cells within the tumor core. They should be microdissected using the 7.5 um laser spot size and the initial power settings recommended by the PixCell II manual. After microdissection all harvested material should immediately be lysed on the cap by applying the lysis buffer (XB) from the PicoPure RNA Isolation Kit according to the PicoPure protocol and stored -80C. Cell populations harvested on different caps can be pooled at the time of RNA isolation. RNA integrity is varified by identification of distinct 28S and 18S ribosomal bands with an Agilent Bioanalyzer using the RNA 6000 Nano LabChip kit. Total RNA was isolated from 2000 LCM cells (to ensure at least 500 ng total RNA) using the PicoPure RNA solation Kit, following manufacturers protocol. mRNA is reverse transcribed with the RiboAmp RNA Amplification kit. 500 ng total RNA is amplified with the RiboAmp RNA Amplification kit, following manufacturerâs instructions. The total yield that can be expected falls between 30 and 60 µg copy RNA. The size of the copy RNA should be verified by gel electrophoresis. Approximately 500ng of copy RNA can be separated on a 1% agarose gel in TAE buffer. A smear of amplified material should be seen between 200 and 3000bp. Six µg amplified RNA are labeled in a RT with SuperScriptIII in the presence of dUTP Cy5 utilizing 6 µg random hexamers as primers. Universal reference RNA is amplified one round in the same manner and labeled with Cy3 dUTP. Labeled cDNA is hybridized overnight onto cDNA microarray. Following hybridization, arrays are washed, scanned and quantitated with the Axon GenePix 4000 microarray reader (Axon Instruments). Gene expression results are analyzed using GeneSpring (Silicon Genetics) software. Intensity dependent normalization is applied, where the ratio is reduced to the residual Lowess fit of the intensity versus ratio curve. The measured intensity of each gene is divided by its reference channel (signal from the universal reference RNA) in each sample. When the reference channel is below 10, the data point is considered uninformative. The ratios (sample over reference) for the tumor core experiments and invasive rim experiments are averaged and compared. Genes that are more than two-fold differentially regulated in the majority of the matched core/invasive rim sets are selected.
2010-05-18 | E-GEOD-12689 | biostudies-arrayexpress
Project description:BackgroundGlioblastoma (GBM) is a primary brain tumor with a 5-year survival rate of ≤5%. We have shown earlier that GBM-antibody-linked curcumin (CC) and also phytosomal curcumin (CCP) rescue 50-60% of GBM-bearing mice while repolarizing the tumor-associated microglia/macrophages (TAM) from the tumor-promoting M2-type to the tumoricidal M1-type. However, systemic application of CCP yields only sub-IC50 concentrations of CC in the plasma, which is unlikely to kill GBM cells directly. This study investigates the role of CC-evoked intra-GBM recruitment of activated natural killer (NK) cells in the elimination of GBM and GBM stem cells.MethodsWe have used an immune-competent syngeneic C57BL6 mouse model with the mouse-GBM GL261 cells orthotopically implanted in the brain. Using immunohistochemistry and flow cytometry, we have quantitatively analyzed the role of the intra-GBM-recruited NK cells by (i) injecting (i.p.) the NK1.1 antibody (NK1.1Ab) to temporarily eliminate the NK cells and (ii) blocking NK recruitment by injecting an IL12 antibody (IL12Ab). The treatment cohorts used randomly-chosen GL261-implanted mice and data sets were compared using two-tailed t-test or ANOVA.ResultsCCP treatment caused the GBM tumor to acquire M1-type macrophages (50-60% of the TAM) and activated NK cells. The treatment also elicited (a) suppression of the M2-linked tumor-promoting proteins STAT3, ARG1, and IL10, (b) induction of the M1-linked anti-tumor proteins STAT1 and inducible nitric oxide synthase in the TAM, (c) elimination of CD133(+) GBM stem cells, and (d) activation of caspase3 in the GBM cells. Eliminating intra-GBM NK cell recruitment caused a partial reversal of each of these effects. Concomitantly, we observed a CCP-evoked dramatic induction of the chemokine monocyte chemotactic protein-1 (MCP-1) in the TAM.ConclusionsThe recruited NK cells mediate a major part of the CCP-evoked elimination of GBM and GBM stem cells and stabilization of the TAM in the M1-like state. MCP-1 is known to activate peripheral M1-type macrophages to secrete IL12, an activator of NK cells. Based on such observations, we postulate that by binding to peripheral M1-type macrophages and IL12-activated NK cells, the brain-released chemokine MCP-1 causes recruitment of peripheral immune cells into the GBM, thereby causing destruction of the GBM cells and GBM stem cells.
| S-EPMC6058381 | biostudies-literature
Project description:Glioblastoma multiforme (GBM), the most advanced form of a large subset of brain tumors collectively known as glioma, is the most aggressive and invasive type of brain tumor. Patients usually experience a median survival range of 9 to 12 months. GBMs are extremely difficult to manage because of the tumor cells’ tendency to migrate and pervade into adjacent tissues. Surgical resection of GBMs generally only slows disease progression because any remaining tumor cells proceed to migrate through brain tissues and reform a new tumor mass. It is hypothesized that the invasive phenotype of these tumor cells may be attributable to unique gene expression. Stationary core and invasive rim tumor cells were collected separately by laser capture microdissection (LCM) from 19 biopsy samples. Identification of differentially expressed genes in the tumor core and invasive rim can give valuable insight to the genes and pathways potentially involved with the invasive phenotype. This information can then be used to generate possible biomarkers, diagnostic markers, or drug targets.
2008-10-19 | GSE12689 | GEO
Project description:Increasing evidence demonstrates that interleukin (IL)-32 is a pro-inflammatory cytokine, inducing IL-1alpha, IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and chemokines via nuclear factor (NF)-kappaB, p38 mitogen-activated protein kinase (MAPK), and activating protein (AP)-1 activation. Here we report that IL-32 is expressed and is also functional in human vascular endothelial cells (EC) of various origins. Compared with primary blood monocytes, high levels of IL-32 are constitutively produced in human umbilical vein EC (HUVEC), aortic macrovascular EC, and cardiac as well as pulmonary microvascular EC. At concentrations as low as 0.1 ng/ml, IL-1beta stimulated IL-32 up to 15-fold over constitutive levels, whereas 10 ng/ml of TNFalpha or 100 ng/ml of lipopolysaccharide (LPS) were required to induce similar quantities of IL-32. IL-1beta-induced IL-32 was reduced by inhibition of the IkappaB kinase-beta/NF-kappaB and ERK pathways. In addition to IL-1beta, pro-coagulant concentrations of thrombin or fresh platelets increased IL-32 protein up to 6-fold. IL-1beta and thrombin induced an isoform-switch in steady-state mRNA levels from IL-32alpha/gamma to beta/epsilon. Adult EC responded in a similar fashion. To prove functionality, we silenced endogenous IL-32 with siRNA, decreasing intracellular IL-32 protein levels by 86%. The knockdown of IL-32 resulted in reduction of constitutive as well as IL-1beta-induced intercellular adhesion molecule-1 (ICAM-1) (of 55% and 54%, respectively), IL-1alpha (of 62% and 43%), IL-6 (of 53% and 43%), and IL-8 (of 46% and 42%). In contrast, the anti-inflammatory/anti-coagulant CD141/thrombomodulin increased markedly when IL-32 was silenced. This study introduces IL-32 as a critical regulator of endothelial function, expanding the properties of this cytokine relevant to coagulation, endothelial inflammation, and atherosclerosis.
| S-EPMC2656174 | biostudies-literature
Project description:ObjectivePreviously we identified palmitoyl-lysophosphatidylcholine (LPC 16:0), as well as linoleoyl-, arachidonoyl- and oleoyl-LPC (LPC 18:2, 20:4 and 18:1) as the most prominent LPC species generated by the action of endothelial lipase (EL) on high-density lipoprotein (HDL). In the present study, the impact of EL and EL-generated LPC on interleukin-8 (IL-8) synthesis was examined in vitro in primary human aortic endothelial cells (HAEC) and in mice.Methods and resultsAdenovirus-mediated overexpression of the catalytically active EL, but not its inactive mutant, increased endothelial synthesis of IL-8 mRNA and protein in a time- and HDL-concentration-dependent manner. While LPC 18:2 was inactive, LPC 16:0, 18:1 and 20:4 promoted IL-8 mRNA- and protein-synthesis, differing in potencies and kinetics. The effects of all tested LPC on IL-8 synthesis were completely abrogated by addition of BSA and chelation of intracellular Ca(2+). Underlying signaling pathways also included NFkB, p38-MAPK, ERK, PKC and PKA. In mice, adenovirus-mediated overexpression of EL caused an elevation in the plasma levels of MIP-2 (murine IL-8 analogue) accompanied by a markedly increased plasma LPC/PC ratio. Intravenously injected LPC also raised MIP-2 plasma concentration, however to a lesser extent than EL overexpression.ConclusionOur results indicate that EL and EL-generated LPC, except of LPC 18:2, promote endothelial IL-8 synthesis, with different efficacy and kinetics, related to acyl-chain length and degree of saturation. Accordingly, due to its capacity to modulate the availability of the pro-inflammatory and pro-adhesive chemokine IL-8, EL should be considered an important player in the development of atherosclerosis.
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Project description:In this study, we investigated the role of cancer-associated fibroblasts (CAFs) in hepatocellular carcinoma (HCC) progression. We showed that CAFs secrete high levels of IL-6, which promoted stem cell-like properties in HCC cells by activating Notch signaling through STAT3 Tyr705 phosphorylation. These effects were abolished by Notch1 shRNA knockdown in HCC cells or treatment with an IL-6 neutralizing antibody or the p-STAT3 (Tyr705) inhibitor cryptotanshinone. Xenografted liver tumors were larger in nude mice injected with HCC cells and CAFs than in those receiving HCC cells alone. Moreover, immunohistochemical analysis of liver tissue specimens from 88 HCC patients revealed that high nuclear Notch intracellular domain (nNICD) levels in HCC cells correlated with poor prognosis in patients. These findings suggest that IL-6 secreted by CAFs promotes stem cell-like properties in HCC cells by enhancing STAT3/Notch signaling.
| S-EPMC5835697 | biostudies-literature