Chimeric antigen receptor (CAR) T-cell therapy is an emerging immunotherapy against several malignancies including glioblastoma, the most common and most aggressive malignant primary brain tumor in adults. The challenges in solid tumor immunotherapy comprise heterogenously expressed tumor target antigens and restricted trafficking of CAR T cells to and impaired long-term persistence at the tumor site, as well as the unaddressed integration of CAR T-cell therapy into conventional anticancer treatments. We addressed these questions using a NKG2D-based chimeric antigen receptor construct (chNKG2D) in fully immunocompetent orthotopic glioblastoma mouse models. ChNKG2D T cells demonstrated high IFNγ production and cytolytic activity in vitro. Upon systemic administration in vivo, chNKG2D T cells migrated to the tumor site in the brain, did not induce adverse events, prolonged survival, and cured a fraction of glioma-bearing mice. Surviving mice were protected long-term against tumor rechallenge. Mechanistically, this was not solely the result of a classical immune memory response, but rather involved local persistence of chNKG2D T cells. A subtherapeutic dose of local radiotherapy in combination with chNKG2D T-cell treatment resulted in synergistic activity in two independent syngeneic mouse glioma models by promoting migration of CAR T cells to the tumor site and increased effector functions. We thus provide preclinical proof-of-concept of NKG2D CAR T-cell activity in mouse glioma models and demonstrate efficacy, long-term persistence, and synergistic activity in combination with radiotherapy, providing a rationale to translate this immunotherapeutic strategy to human glioma patients. Significance: These findings provide evidence for synergy of conventional anticancer therapy and CAR T cells and heralds future studies for other treatment combinations. Cancer Res; 78(4); 1031–43. ©2017 AACR.

J. Neurochem. (2011) 118, 449–457. The prognosis of patients afflicted by glioblastoma remains poor. Biomarkers for the disease would be desirable in order to allow for an early detection of tumor progression or to indicate rapidly growing tumor subtypes requiring more intensive therapy. In this study, we investigated whether a blood‐derived specific miRNA fingerprint can be defined in patients with glioblastoma. To this end, miRNA profiles from the blood of 20 patients with glioblastoma and 20 age‐ and sex‐matched healthy controls were compared. Of 1158 tested miRNAs, 52 were significantly deregulated, as assessed by unadjusted Student′s t‐test at an alpha level of 0.05. Of these, two candidates, miR‐128 (up‐regulated) and miR‐342‐3p (down‐regulated), remained significant after correcting for multiple testing by Benjamini–Hochberg adjustment with a p‐value of 0.025. The altered expression of these two biomarkers was confirmed in a second cohort of glioblastoma patients and healthy controls by real‐time PCR and validated for patients who had received neither radio‐ nor chemotherapy and for patients who had their glioblastomas resected more than 6 months ago. Moreover, using machine learning, a comprehensive miRNA signature was obtained that allowed for the discrimination between blood samples of glioblastoma patients and healthy controls with an accuracy of 81% [95% confidence interval (CI) 78–84%], specificity of 79% (95% CI 75–83%) and sensitivity of 83% (95% CI 71–85%). In summary, our proof‐of‐concept study demonstrates that blood‐derived glioblastoma‐associated characteristic miRNA fingerprints may be suitable biomarkers and warrant further exploration.

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.

Immunocytokines display promising antitumor activity in orthotopic mouse models of glioma and patients with glioblastoma.

Purpose: NKG2D is a potent activating immune cell receptor, and glioma cells express the cognate ligands (NKG2DL). These ligands are inducible by cellular stress and temozolomide (TMZ) or irradiation (IR), the standard treatment of glioblastoma, could affect their expression. However, a role of NKG2DL for the efficacy of TMZ and IR has never been addressed. Experimental Design: We assessed the effect of TMZ and IR on NKG2DL in vitro and in vivo in a variety of murine and human glioblastoma models, including glioma-initiating cells, and a cohort of paired glioblastoma samples from patients before and after therapy. Functional effects were studied with immune cell assays. The relevance of the NKG2D system for the efficacy of TMZ and IR was assessed in vivo in syngeneic orthotopic glioblastoma models with blocking antibodies and NKG2D knockout mice. Results: TMZ or IR induced NKG2DL in vitro and in vivo in all glioblastoma models, and glioblastoma patient samples had increased levels of NKG2DL after therapy with TMZ and IR. This enhanced the immunogenicity of glioma cells in a NGK2D-dependent manner, was independent from cytotoxic or growth inhibitory effects, attenuated by O⁶-methylguanine-DNA-methyltransferase (MGMT), and required the DNA damage response. The survival benefit afforded by TMZ or IR relied on an intact NKG2D system and was decreased upon inhibition of the NKG2D pathway. Conclusions: The immune system may influence the activity of convential cancer treatments with particular importance of the NKG2D pathway in glioblastoma. Our data provide a rationale to combine NKG2D-based immunotherapies with TMZ and IR. Clin Cancer Res; 24(4); 882–95. ©2017 AACR.

Glioblastoma is the most common and aggressive form of intrinsic brain tumor with a very poor prognosis. Thus, novel therapeutic approaches are urgently needed. Tumor-treating fields (TTFields) may represent such a novel treatment option. The aim of this study was to investigate the effects of TTFields on glioma cells, as well as the functional characterization of the underlying mechanisms. Here, we assessed the anti-glioma activity of TTFields in several preclinical models. Applying TTFields resulted in the induction of cell death in a frequency- and intensity-dependent manner in long-term glioma cell lines, as well as glioma-initiating cells. Cell death occurred in the absence of caspase activation, but involved autophagy and necroptosis. Severe alterations in cell cycle progression and aberrant mitotic features, such as poly- and micronucleation, preceded the induction of cell death. Furthermore, exposure to TTFields led to reduced migration and invasion, which are both biological hallmarks of glioma cells. The combination of TTFields with irradiation or the alkylating agent, temozolomide (TMZ), resulted in additive or synergistic effects, and the O⁶-methyl-guanine DNA methyltransferase status did not influence the efficacy of TTFields. Importantly, TMZ-resistant glioma cells were responsive to TTFields application, highlighting the clinical potential of this therapeutic approach. In summary, our results indicate that TTFields induce autophagy, as well as necroptosis and hamper the migration and invasiveness of glioma cells. These findings may allow for a more detailed clinical evaluation of TTFields beyond the clinical data available so far.