The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-γ and TNF-α, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-γ and TNF-α synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

Glioblastoma multiforme (GBM) is the most frequent and aggressive brain tumor in adults. The dogma that GBM spread is restricted to the brain was challenged by reports on extracranial metastases after organ transplantation from GBM donors. We identified circulating tumor cells (CTCs) in peripheral blood (PB) from 29 of 141 (20.6%) GBM patients by immunostaining of enriched mononuclear cells with antibodies directed against glial fibrillary acidic protein (GFAP). Tumor cell spread was not significantly enhanced by surgical intervention. The tumor nature of GFAP-positive cells was supported by the absence of those cells in healthy volunteers and the presence of tumor-specific aberrations such as EGFR gene amplification and gains and losses in genomic regions of chromosomes 7 and 10. Release of CTCs was associated with EGFR gene amplification, suggesting a growth potential of these cells. We demonstrate that hematogenous GBM spread is an intrinsic feature of GBM biology.

NK cells are promising effector cells for adjuvant immunotherapy of cancer. So far, several preclinical studies have shown the feasibility of gene-engineered NK cells, which upon expression of chimeric antigen receptors (CARs) are redirected to otherwise NK-cell resistant tumors. Yet, we reasoned that the efficiency of an immunotherapy using CAR-modified NK cells critically relies on efficient migration to the tumor site and might be improved by the engraftment of a receptor specific for a chemokine released by the tumor. Based on the DNAX-activation protein 12 (DAP12), a signaling adapter molecule involved in signal transduction of activating NK cell receptors, we constructed an EGFRvIII-CAR, designated MR1.1-DAP12 which confers specific cytotoxicity of NK cell towards EGFRvIII+ glioblastoma cells in vitro and to established subcutaneous U87-MGEGFRvIII tumor xenografts. So far, infusion of NK cells with expression of MR1.1-DAP12 caused a moderate but significantly delayed tumor growth and increased median survival time when compared to NK cells transduced with an ITAM-defective CAR. Notably, the further genetic engineering of these EGFRvIII-specific NK cells with the chemokine receptor CXCR4 conferred a specific chemotaxis to CXCL12/SDF-1α secreting U87-MG glioblastoma cells. Moreover, the administration of such NK cells resulted in complete tumor remission in a number of mice and a significantly increased survival when compared to the treatment of xenografts with NK cells expressing only the EGFRvIII-specific CAR or mock control. We conclude that chemokine receptor engineered NK cells with concomitant expression of a tumor-specific CAR are a promising tool to improve adoptive tumor immunotherapy.

Glioblastomas contain stem-like cells that can be maintained in vitro using specific serum-free conditions. We investigated whether glioblastoma stem-like (GS) cell lines preserve the expression phenotype of human glioblastomas more closely than conventional glioma cell lines. Expression profiling revealed that a distinct subset of GS lines, which displayed a full stem-like phenotype (GSf), mirrored the expression signature of glioblastomas more closely than either other GS lines or cell lines grown in serum. GSf lines are highly tumorigenic and invasive in vivo, express CD133, grow spherically in vitro, are multipotent and display a Proneural gene expression signature, thus recapitulating key functional and transcriptional aspects of human glioblastomas. In contrast, GS lines with a restricted stem-like phenotype exhibited expression signatures more similar to conventional cell lines than to original patient tumors, suggesting that the transcriptional resemblance between GS lines and tumors is associated with different degrees of "stemness". Among markers overexpressed in patient tumors and GSf lines, we identified CXCR4 as a potential therapeutic target. GSf lines contained a minor population of CXCR4(hi) cells, a subfraction of which coexpressed CD133 and was expandable by hypoxia, whereas conventional cell lines contained only CXCR4(lo) cells. Convection-enhanced local treatment with AMD3100, a specific CXCR4 antagonist, inhibited the highly invasive growth of GS xenografts in vivo and cell migration in vitro. We thus demonstrate the utility of GSf lines in testing therapeutic agents and validate CXCR4 as a target to block the growth of invasive tumor-initiating glioma stem cells in vivo.

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.

The transmembrane chemokine CXCL16 is expressed by dendritic and vascular cells and mediates chemotaxis and adhesion of activated T cells via the chemokine receptor CXCR6/Bonzo. Here we describe the expression and shedding of this chemokine by glioma cells in situ and in vitro. By quantitative RT-PCR and immunohistochemistry, we show that CXCL16 is highly expressed in human gliomas, while expression in normal brain is low and mainly restricted to brain vascular endothelial cells. In cultivated human glioma cells as well as in activated mouse astroglial cells, CXCL16 mRNA and protein is constitutively expressed and further up-regulated by tumour necrosis factor a (TNFa) and interferon-c (IFNc). CXCL16 is continuously released from glial cells by proteolytic cleavage which is rapidly enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). As shown by inhibitor studies, two distinct members of the disintegrin-like metalloproteinase family ADAM10 and 17 are involved in the constitutive and PMA-induced shedding of glial CXCL16. In addition to the chemokine, its receptor CXCR6 could be detected by quantitative RT-PCR in human glioma tissue, cultivated murine astrocytes and at a lower level in microglial cells. Functionally, recombinant soluble CXCL16 enhanced proliferation of CXCR6-positive murine astroglial and microglial cells. Thus, the transmembrane chemokine CXCL16 is expressed in the brain by malignant and inflamed astroglial cells, shed to a soluble form and targets not only activated T cells but also glial cells themselves.

Cetuximab and panitumumab, two antibodies targeting the extracellular domain of the epidermal growth factor receptor (EGFR), are of major clinical importance particularly in the treatment of metastatic colorectal cancer. As patients may acquire resistance-mediating mutations within the extracellular EGFR domain, functional dissection of the exact binding sites of EGFR targeting antibodies may help predict treatment responses. We therefore assessed the epitope recognition of panitumumab by screening phage-displayed random cyclic 7mer and linear 12mer peptide libraries on this antibody. Phage screenings revealed two strong, potentially epitope-mimicking consensus motifs targeted by panitumumab. A computational approach was used to map the sequences back to the potential epitope region on domain III of EGFR. The presumed epitope regions (386)WPEXRT(391) and a biochemically similar though discontinuous region P349-F352-D355 on a neighboring loop of domain III could be confirmed as part of the functionally relevant binding site of panitumumab by site-directed mutational analysis. To more accurately differentiate the panitumumab epitope from the previously characterized cetuximab epitope, binding studies were performed on a broad range of additional mutants. Taken together, this analysis revealed two large, partially overlapping functional epitopes consisting of 17 critical amino acid positions. Four of these positions were selectively targeted by cetuximab (I467, S468, Q408, and H409), whereas another four were selectively recognized by panitumumab (W386, E388, R390, and T391). In view of the clinical significance of extracellular domain mutations, our data may help guide treatment decisions in selected patients receiving EGFR-targeted therapies.

Fluctuations in oxygen tension during tissue remodeling impose a major metabolic challenge in human tumors. Stem-like tumor cells in glioblastoma, the most common malignant brain tumor, possess extraordinary metabolic flexibility, enabling them to initiate growth even under non-permissive conditions. We identified a reciprocal metabolic switch between the pentose phosphate pathway (PPP) and glycolysis in glioblastoma stem-like (GS) cells. Expression of PPP enzymes is upregulated by acute oxygenation but downregulated by hypoxia, whereas glycolysis enzymes, particularly those of the preparatory phase, are regulated inversely. Glucose flux through the PPP is reduced under hypoxia in favor of flux through glycolysis. PPP enzyme expression is elevated in human glioblastomas compared to normal brain, especially in highly proliferative tumor regions, whereas expression of parallel preparatory phase glycolysis enzymes is reduced in glioblastomas, except for strong upregulation in severely hypoxic regions. Hypoxia stimulates GS cell migration but reduces proliferation, whereas oxygenation has opposite effects, linking the metabolic switch to the "go or grow" potential of the cells. Our findings extend Warburg's observation that tumor cells predominantly utilize glycolysis for energy production, by suggesting that PPP activity is elevated in rapidly proliferating tumor cells but suppressed by acute severe hypoxic stress, favoring glycolysis and migration to protect cells against hypoxic cell damage.