Isabel Tritschler

Toll‐Like Receptor Engagement Enhances the Immunosuppressive Properties of Human Bone Marrow‐Derived Mesenchymal Stem Cells by Inducing Indoleamine‐2,3‐dioxygenase‐1 via Interferon‐β and Protein Kinase R

Mesenchymal stem cells (MSC) display unique suppressive properties on T‐cell immunity, thus representing an attractive vehicle for the treatment of conditions associated with harmful T‐cell responses such as organ‐specific autoimmunity and graft‐versus‐host disease. Toll‐like receptors (TLR) are primarily expressed on antigen‐presenting cells and recognize conserved pathogen‐derived components. Ligation of TLR activates multiple innate and adaptive immune response pathways to eliminate and protect against invading pathogens. In this work, we show that TLR expressed on human bone marrow‐derived MSC enhanced the immunosuppressive phenotype of MSC. Immunosuppression mediated by TLR was dependent on the production of immunosuppressive kynurenines by the tryptophan‐degrading enzyme indoleamine‐2,3‐dioxygenase‐1 (IDO1). Induction of IDO1 by TLR involved an autocrine interferon (IFN)‐β signaling loop, which was dependent on protein kinase R (PKR), but independent of IFN‐γ. These data define a new role for TLR in MSC immunobiology, which is to augment the immunosuppressive properties of MSC in the absence of IFN‐γ rather than inducing proinflammatory immune response pathways. PKR and IFN‐β play a central, previously unidentified role in orchestrating the production of immunosuppressive kynurenines by MSC. STEM CELLS 2009;27:909–919

Cilengitide modulates attachment and viability of human glioma cells, but not sensitivity to irradiation or temozolomide in vitro

Cilengitide is a cyclic peptide antagonist of integrins alphavbeta3 and alphavbeta5 that is currently being evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic but may include direct effects on tumor cells, notably on attachment, migration, invasion, and viability. In this study we found that, at clinically relevant concentrations, cilengitide (1-100 microM) induces detachment in some but not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by cilengitide could not be predicted by the level of expression of the cilengitide target molecules, alphavbeta3 and alphavbeta5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was not required for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad-spectrum caspase inhibitor zVAD-fmk was not protective. Moreover, forced expression of the antiapoptotic protein marker Bcl-X(L) or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered glioma cell responses to cilengitide alone or to cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion, which promotes temozolomide delivery to glioma cells.

GDF-15 Contributes to Proliferation and Immune Escape of Malignant Gliomas

Purpose: Growth and differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-β family. GDF-15 is necessary for the maintenance of pregnancy but has also been linked to other physiologic and pathologic conditions. Experimental Design: The expression of GDF-15 in glioma cell lines was assessed by quantitative reverse transcriptase-PCR and immunoblot. GDF-15 levels in situ and in the peripheral blood of glioma patients were examined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The effects of short hairpin RNA-mediated GDF-15 inhibition on proliferation and immunogenicity of SMA-560 glioma cells were investigated by [methyl-3H]thymidine incorporation and immune-mediated target cell lysis. The impact of GDF-15 on glioma growth in vivo was assessed in syngeneic mice. Results: GDF-15 is expressed by gliomas of different WHO grades as assessed by immunohistochemistry. The high expression of GDF-15 in tumor tissue translates into elevated GDF-15 serum levels in glioblastoma patients compared with healthy controls. GDF-15 mRNA and protein are also detectable in human and mouse glioma cells in vitro. Silencing of GDF-15 by RNA interference reduces the proliferation of malignant glioma cells. Immunologically, the depletion of glioma-derived GDF-15 enhances the susceptibility of mouse glioma cells towards syngeneic natural killer cells and splenocytes. This results in a reduced in vivo tumorigenicity and increased T-cell infiltration of GDF-15–deficient glioma cells in syngeneic mice. Conclusions: Although previous studies focusing on ectopic overexpression of GDF-15 have proposed unclear or antitumorigenic effects of GDF-15 in glioma cells, we here show that GDF-15 at endogenous levels contributes to proliferation and immune escape of malignant gliomas in an immunocompetent host. Clin Cancer Res; 16(15); 3851–9. ©2010 AACR.

Aryl hydrocarbon receptor inhibition downregulates the TGF-Β/Smad pathway in human glioblastoma cells

The dioxin/aryl hydrocarbon receptor (AhR) is a transcription factor, which has been attributed a role in human cancerogenesis, cell cycle progression and transforming growth factor-β (TGF-β) signaling. As TGF-β is an important mediator of the malignant phenotype of human gliomas, we studied AhR expression and function in glioma cells. AhR was not only expressed in glioma cells in vitro, but was also detected in human gliomas in vivo by immunohistochemistry, with a predominantly nuclear staining in glioblastomas. The AhR agonist, 3-methylcholanthrene, induced AhR nuclear translocation and upregulated mRNA levels of the AhR target gene, cytochrome P450 1A1 (CYP1A1). Conversely, pharmacological inhibition of AhR using the novel AhR antagonist, CH-223191, or AhR gene silencing using small interfering RNA showed that constitutive AhR activity positively controls TGF-β1, TGF-β2 and latent TGF-β-binding protein-1 protein levels in malignant glioma cells. Moreover, antagonism of AhR reduced clonogenic survival and invasiveness of glioma cells. In contrast, AhR regulates TGF-β signaling negatively in non-neoplastic astrocytes. Thus, the pathogenesis of glioma formation may involve altered AhR regulation of the TGF-β/Smad pathway, and AhR may represent a promising target for the treatment of human malignant gliomas and other diseases associated with pathological TGF-β activity.

Phosphoglycerate Kinase 1 a Promoting Enzyme for Peritoneal Dissemination in Gastric Cancer

Peritoneal carcinomatosis is a frequent finding in gastric cancer associated with a poor prognosis. The features that enable gastric tumors to disseminate are poorly understood until now. Previously, we showed elevated mRNA levels of phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate‐generating enzyme in the glycolytic pathway, the chemokine receptor 4 (CXCR4), the corresponding chemokine ligand 12 (CXCL12) and β‐catenin in specimens from gastric cancer patients with peritoneal carcinomatosis. In this study, the influence of PGK1 on CXCR4 and β‐catenin was assessed as well as the invasiveness of PGK1 overexpressing cancer cells. In this current study, we found that PGK1 regulates the expression of CXCR4 and β‐catenin at the mRNA and protein levels. On the other hand, CXCR4 regulates the expression of PGK1. Plasmid‐mediated overexpression of PGK1 dramatically increased the invasiveness of gastric cancer cells. Interestingly, inhibition of CXCR4 in cells overexpressing PGK1 produced only a moderate reduction of invasiveness suggesting that, PGK1 itself has a critical role in tumor invasiveness. Immunohistochemistry in specimens from diffuse gastric cancer patients also revealed an overexpression of PGK1 in patients with development of peritoneal carcinomatosis. Therefore, PGK1 may be a crucial enzyme in peritoneal dissemination. Together these findings suggest that the enhanced expression of PGK1 and its signaling targets CXCR4 and β‐catenin in gastric cancer cells promote peritoneal carcinomatosis. Thus, PGK1 may serve as prognostic marker and/or be a potential therapeutic target to prevent dissemination of gastric carcinoma cells into the peritoneum.

Modulation of TGF-β activity by latent TGF-β-binding protein 1 in human malignant glioma cells

High biological activity of the transforming growth factor (TGF)‐β‐Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF‐β has become a novel target for the experimental treatment of these tumors. TGF‐β is processed by furin‐like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency‐associated peptide (LAP). Latent TGF‐β‐binding protein 1 (LTBP‐1) covalently binds to this small latent TGF‐β complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP‐1 protein in vivo increase with the grade of malignancy in gliomas. LTBP‐1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP‐1 into the medium is decreased by the inhibition of FLP activity. Gene‐transfer mediated overexpression of LTBP‐1 in glioma cell lines results in an increase inTGF‐β activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF‐β activity is enhanced. Conversely, LTBP‐1 gene silencing reduces TGF‐β activity and Smad2 phosphorylation without affecting TGF‐β protein levels. Collectively, we identify LTBP‐1 as an important modulator of TGF‐β activation in glioma cells, which may contribute to the malignant phenotype of these tumors.

A disintegrin and metalloproteinases 10 and 17 modulate the immunogenicity of glioblastoma-initiating cells

BACKGROUND There are emerging reports that the family of a disintegrin and metalloproteinases (ADAM) are involved in the maintenance of the malignant phenotype of glioblastomas. Notably, ADAM proteases 10 and 17 might impair the immune recognition of glioma cells via the activating immunoreceptor NKG2D by cleavage of its ligands from the cell surface. Glioblastoma-initiating cells (GIC) with stem cell properties have been identified as an attractive target for immunotherapy. However, GIC immunogenicity seems to be low. METHODS AND RESULTS Here,we show that ADAM10 and ADAM17 are expressed on the cell surface of GIC and contribute to an immunosuppressive phenotype by cleavage of ULBP2. The cell surface expression of ULBP2 is enhanced upon blocking ADAM10 and ADAM17, and treatment with ADAM10 and ADAM17specific inhibitors leads to enhanced immunerecognition of GIC by natural killer cells. CONCLUSIONS Therefore, ADAM10 and ADAM17 constitute suitable targets to boost an immune response against GIC.