Kirsten Hattermann

The presumed atypical chemokine receptor CXCR7 signals through Gi/o proteins in primary rodent astrocytes and human glioma cells

SDF‐1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF‐1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin‐sensitive Gi/o proteins. SDF‐1‐dependent activation of Gi/o proteins and subsequent increases in intracellular Ca2+ concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7‐mediated effects of SDF‐1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF‐1‐induced activation of Erk and Akt in CXCR7‐only expressing human glioma cell lines. Finally, consistent with a ligand‐biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I‐TAC/CXCL11, activated Erk and Akt through β‐arrestin. The demonstration that SDF‐1‐bound CXCR7 activates Gi/o proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.

CX3CR1 promotes recruitment of human glioma-infiltrating microglia/macrophages (GIMs)

The transmembrane chemokine CX3CL1 and its receptor CX3CR1 are thought to be involved in the trafficking of immune cells during an immune response and in the pathology of various human diseases including cancer. However, little is known about the expression and function of CX3CR1 in human glioma-infiltrating microglia/macrophages (GIMs), representing the major cellular stroma component of highly malignant gliomas. Here, we show that CX3CR1 is overexpressed at both the mRNA and protein level in solid human astrocytomas of different malignancy grades and in glioblastomas. CX3CR1 was localized in ionized calcium-binding adapter molecule 1 (Iba1) and CD11b/c positive GIMs in situ as shown by fluorescence microscopy. In accordance with this, freshly isolated human GIM-enriched fractions separated by CD11b MACS technology displayed high Iba1 and CX3CR1 mRNA expression levels in vitro. Moreover, cultured human GIMs responded to CX3CL1-triggered activation of CX3CR1 with adhesion and migration in vitro. Besides an increase in motility, CX3CL1 also enhanced expression of matrix metalloproteases 2, 9, and 14 in GIM fractions in vitro. These data indicate that the CX3CL1/CX3CR1 system has a crucial tumor-promoting role in human glioblastomas via its impact on glioma-infiltrating immune subsets.

Endoglin expression in metastatic breast cancer cells enhances their invasive phenotype

Endoglin is a cell-surface adhesion protein as well as a coreceptor for transforming growth factor-β (TGF-β). It is located on endothelial and few other cells, but also found on certain tumor cells. Brain metastatic breast tumor cells derived from the MDA-MB-231 cell line heavily express endoglin in contrast to the corresponding parental ones. To clarify whether this determines their invasive phenotype, we compared their biological properties with endoglin-silenced brain-metastatic cells, low-expressing parental cells and these transfected with L- and S-endoglins, isoforms transducing or lacking TGF-β signals. All L-endoglin-overexpressing cells were characterized by numerous invadopodia where endoglin was preferentially localized. Endoglin-expression resulted in elevated levels of the matrix metalloproteinases (MMP-1 and MMP-19) and downregulation of the plasminogen activator inhibitor-1. In Boyden-chamber and wound-healing assays, endoglin-overexpressing cells showed a considerably higher migration and chemotaxis to TGF-β. In 3D spheroid confrontation assays between breast tumor cells and TGF-β-secreting glioma cells, high L-endoglin-expressing cells invaded into the glioma-spheroids whereas low-endoglin-expressing cells dissociated in the culture; invasion was blocked by TGF-β antibodies. In contrast to parental cells, endoglin-overexpressing cells invaded deeply into mouse brain slices. Thus, endoglin expression on tumor cells enhances their invasive character by formation of invadopodia, extracellular proteolysis, chemotaxis and migration.

An Infernal Trio: The chemokine CXCL12 and its receptors CXCR4 and CXCR7 in tumor biology

Chemokines are small peptide mediators that play a role in many physiological and pathological processes. Apart from their initially discovered function in trafficking of leukocytes, they also influence migration, proliferation, survival and gene expression of a variety of cell types in their respective microenvironment. Chemokines can exert these effects via their respective G protein-coupled receptor. Over the recent decade, the involvement of chemokines and their respective receptors in tumor biology has been successively elucidated. This review will focus on the signaling and effects of the widespread chemokine CXCL12 and its long known G protein-coupled receptor CXCR4 and the recently discovered non-G protein-coupled receptor CXCR7 with a detailed reflection on glioma biology.

Lost in disruption: role of proteases in glioma invasion and progression.

A characteristic feature of malignant glial tumors (gliomas) is their tendency to diffusely infiltrate the nervous system preventing their complete surgical resection. Proteases play a decisive role in this malignant process, either by degradation of brain extracellular matrix (ECM) components, adhesion molecules, or by regulating the activity of growth and chemotactic factors. Secreted matrix metalloproteinases (MMPs) and ADAMTS proteases (ADAMs with thrombospondin motifs) cleave different ECM components like the proteoglycans (lecticans) aggrecan, versican, neurocan and brevican with selective preferences; they are further regulated by endogenous inhibitors and activating metallo- and serine proteases. Cell surface proteases of the ADAM family (A Disintegrin And Metalloproteinase), but also serine proteases regulate the activity of growth factors and chemokines that act as autocrine / paracrine stimulators within gliomas. Thus, proteases play a decisive role for the spread and growth of gliomas and are prominent targets for their therapy.

The chemokine CXCL16 induces migration and invasion of glial precursor cells via its receptor CXCR6.

Chemokines are implicated in developmental and inflammatory processes in the brain. The transmembrane chemokine CXCL16 is produced in brain endothelial and reactive astroglial cells and released by shedding. Its receptor CXCR6 is detected during brain development highest at postnatal day 6, found in glial precursor cells differentiated from neural stem cells and in an A2B5-positive glial precursor cell line. Their stimulation by soluble CXCL16 induces the PI3-kinase/Akt and Erk pathways resulting in the activation of the transcription factor AP-1. As biological responses, soluble CXCL16 upregulates its own receptor, increases cell proliferation, stimulates cell migration in wound-healing and in spheroid confrontation assays. Invasion of CXCR6-positive glial cells into CXCL16-expressing spheroids can be blocked by sheddase inhibitors and CXCL16-antibody. Since CXCL16 is induced by cytokines at sites of inflammation, neurodegeneration, ischemia and malignant transformation, it should attract CXCR6-positive glial precursor cells, enhance their invasion and proliferation and thus favor astrogliosis.

A methylation-specific and SYBR-green-based quantitative polymerase chain reaction technique for O6-methylguanine DNA methyltransferase promoter methylation analysis

The O(6)-methylguanine DNA methyltransferase (MGMT) gene encodes a DNA repair enzyme whose activity is a major mechanism of resistance to alkylating drugs in glioblastoma treatment. Hypermethylation of the MGMT promoter is associated with chemosensitivity because it reduces MGMT activity. Here we present a method combining methylation-specific and SYBR-green-based quantitative PCR (MSQP) for MGMT promoter methylation analysis. This highly specific, sensitive, and reproducible method allows the quantification of fully methylated and fully unmethylated MGMT DNA species in terms of percentage. Values are related to standard curves, corrected for DNA input by an internal standard, and calculated in relation to methylated and unmethylated control DNAs as a percentage share. Finally, values are defined relative to the sum of fully methylated and unmethylated MGMT DNA sample amount to obtain percentage of methylated reference and percentage of unmethylated reference results. We have used this technique to investigate MGMT promoter methylation in relation to MGMT mRNA expression in nine tumor cell lines and 15 primary glioblastoma patients. Presented data confirm that this assay is suitable for detection of low amounts of methylated and unmethylated MGMT promoter DNA. Carefully validated quantitative MSQP assays will be useful in both research and clinical molecular diagnosis.

Overexpression of CXCL16 and its receptor CXCR6/Bonzo promotes growth of human schwannomas.

Chemokines and their receptors play a decisive role in tumor progression and metastasis. Here, we describe the expression of the CXCL16‐CXCR6‐system in human schwannomas of different localization and in malignant peripheral nerve sheath tumors. The transmembrane chemokine CXCL16 and its receptor CXCR6/Bonzo were overexpressed on the mRNA and protein levels in all tumor samples investigated as compared with normal peripheral or 8th cranial nerve tissues. Chromogenic immunostaining and confocal laser microscopy revealed that CXCL16 and CXCR6 were localized mainly on S‐100 positive schwannoma cells. Cultured schwannoma cells responded to CXCL16‐stimulation by phosphorylation of kinases p42/44 (Erk 2/1) that could be inhibited by the MEK1/2‐inhibitor U0126 indicating an involvement of the mitogen‐activated protein kinase signal transduction pathway. As a biological response, CXCL16 increased proliferation and induced migration of schwannomas. Hence, CXCL16 appears to be a novel growth factor for schwannomas of different localization.

Matrix Metalloproteinase-19 is Highly Expressed in Astroglial Tumors and Promotes Invasion of Glioma Cells

Glial tumors exhibit a high morbidity and mortality because of their invasive nature. Matrix metalloproteinase 19 (MMP19) is a secreted protease that together with epilysin (MMP28) forms a structural subgroup of MMPs. We analyzed their expression by quantitative reverse transcription polymerase chain reaction, Western blot, and immunohistochemistry in tumor and normal control brain tissues and in glioblastoma (GB) cells and performed MMP19 silencing functional assays. Matrix metalloproteinase 28 was transcribed to the same extent in normal brain samples and gliomas but was undetectable in GB cell lines. In contrast, MMP19 was detected by immunohistochemistry in normal brain samples only in endothelial cells but was found at high levels in astrocytomas of different World Health Organization grades in situ and in GB cells in vitro. Matrix metalloproteinase 19 was upregulated in GB cells after exposure to proinflammatory cytokines. In Transwell invasion assays, MMP19-silenced cells migrated more slowly through laminin-, basal lamina-, and brevican-coated membranes than controls. Matrix metalloproteinase 19-silenced GB cells also migrated into brain tissue slices compared with control cells. Brevican, a brain-specific proteoglycan and major component of brain extracellular matrix, was degraded by recombinant human MMP19. Taken together, these results indicate that MMP19 is highly expressed in proliferating astrocytoma/glioma cells, and that its expression may facilitate their invasion through brain extracellular matrix components.

Spheroid confrontation assay: a simple method to monitor the three-dimensional migration of different cell types in vitro.

Cell migration and tissue invasion is an important issue in tumour and developmental research. Here, we describe the spheroid invasion assay in detail, a method for monitoring migration of different cell types in a three-dimensional model in vitro. Different (or same) cell types are fluorescently labelled with two different dyes, e.g. Vybrant® CFDA SE Kit ([5(6)carboxyfluorescein diacetate] succidimidylester, green) or SNARF-1® (red), and spheroids of these cells are formed in a medium with 0.24% methylcellulose. After this, spheroids are detached, picked and confronted with each other. After different periods of time, cell invasion can be easily followed microscopically. As an example, the method was applied here to visualize the migration and invasion of microglial and glial precursor cells into spheroids of tumour cells driven by chemokines or chemotactic growth factors. Antibodies to chemokines or chemotactic growth factors/receptors or inhibitors of signal transduction/proteases generating soluble factors can be used to demonstrate the specificity of the chemotactic agents. Thus, this method provides an easy in vitro-method to monitor chemotaxis and three-dimensional cell migration.