Translocator protein (18kDA) (TSPO) marks mesenchymal glioblastoma cell populations characterized by elevated numbers of tumor-associated macrophages.

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.

Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype.

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma.

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance.

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells.

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo.

Metabolic Heterogeneity of Brain Tumor Cells of Proneural and Mesenchymal Origin.

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors.

Neutrophils Promote Glioblastoma Tumor Cell Migration after Biopsy.

Glioblastoma is diagnosed by biopsy or, if clinically feasible, tumor resection. However, emerging evidence suggests that this surgical intervention may increase the risk of tumor cell spread. It has been hypothesized that the damage to the tumor leads to infiltration of immune cells that consequently form an environment that favors tumor cell motility. In mouse glioma models, it was previously found that biopsy induced migration of tumor cells in vivo and that recruitment of monocytes from the blood was involved in this effect. However, the role of neutrophils in this process is still unclear. Here, we study the contribution of neutrophils on the pro-migratory effect of surgical interventions in glioma. Using repetitive intravital microscopy, in vivo migration of glioma tumor cells before and after biopsy was compared in mice systemically depleted of neutrophils. Interestingly, macrophages/microglia were almost completely absent from neutrophil-depleted tumors, indicating that neutrophils may be indirectly involved in biopsy-induced migration of glioma tumor cells through the recruitment of macrophages to the tumor. To further investigate whether neutrophils have the potential to also directly promote glioblastoma tumor cell migration, we performed in vitro migration assays using human neutrophils. Indeed, wound-healing of human primary glioblastoma tumor cell lines was promoted by human neutrophils. The pro-migratory effects of human neutrophils on glioblastoma tumor cells could also be recapitulated in transwell migration assays, indicating that soluble factor(s) are involved. We therefore provide evidence for both an indirect and direct involvement of neutrophils in tumor spread following biopsy of glioblastoma tumors.

Glioblastoma Invasiveness and Collagen Secretion Are Enhanced by Vitamin C.

Aims: Glioblastoma (GB) is one of the most aggressive brain tumors. These tumors modify their metabolism, increasing the expression of glucose transporters, GLUTs, which incorporate glucose and the oxidized form of vitamin C, dehydroascorbic acid (DHA). We hypothesized that GB cells preferentially take up DHA, which is intracellularly reduced and compartmentalized into the endoplasmic reticulum (ER), promoting collagen biosynthesis and an aggressive phenotype. Results: Our results showed that GB cells take up DHA using GLUT1, while GLUT3 and sodium-dependent vitamin C transporter 2 (SVCT2) are preferably intracellular. Using a baculoviral system and reticulum-enriched extracts, we determined that SVCT2 is mainly located in the ER and corresponds to a short isoform. Ascorbic acid (AA) was compartmentalized, stimulating collagen IV secretion and increasing in vitro and in situ cell migration. Finally, orthotopic xenografts induced in immunocompetent guinea pigs showed that vitamin C deficiency retained collagen, reduced blood vessel invasion, and affected glomeruloid vasculature formation, all pathological conditions associated with malignancy. Innovation and Conclusion: We propose a functional role for vitamin C in GB development and progression. Vitamin C is incorporated into the ER of GB cells, where it favors the synthesis of collagen, thus impacting tumor development. Collagen secreted by tumor cells favors the formation of the glomeruloid vasculature and enhances perivascular invasion. Antioxid. Redox Signal. 37, 538-559.

The Role of Translocator Protein TSPO in Hallmarks of Glioblastoma.

Glioblastoma (GBM) is the most fatal primary brain cancer in adults. Despite extensive treatment, tumors inevitably recur, leading to an average survival time shorter than 1.5 years. The 18 kDa translocator protein (TSPO) is abundantly expressed throughout the body including the central nervous system. The expression of TSPO increases in states of inflammation and brain injury due to microglia activation. Not least due to its location in the outer mitochondrial membrane, TSPO has been implicated with a broad spectrum of functions. These include the regulation of proliferation, apoptosis, migration, as well as mitochondrial functions such as mitochondrial respiration and oxidative stress regulation. TSPO is frequently overexpressed in GBM. Its expression level has been positively correlated to WHO grade, glioma cell proliferation, and poor prognosis of patients. Several lines of evidence indicate that TSPO plays a functional part in glioma hallmark features such as resistance to apoptosis, invasiveness, and proliferation. This review provides a critical overview of how TSPO could regulate several aspects of tumorigenesis in GBM, particularly in the context of the hallmarks of cancer proposed by Hanahan and Weinberg in 2011.

Tumor Cell Invasion in Glioblastoma.

Glioblastoma (GBM) is a particularly devastating tumor with a median survival of about 16 months. Recent research has revealed novel insights into the outstanding heterogeneity of this type of brain cancer. However, all GBM subtypes share the hallmark feature of aggressive invasion into the surrounding tissue. Invasive glioblastoma cells escape surgery and focal therapies and thus represent a major obstacle for curative therapy. This review aims to provide a comprehensive understanding of glioma invasion mechanisms with respect to tumor-cell-intrinsic properties as well as cues provided by the microenvironment. We discuss genetic programs that may influence the dissemination and plasticity of GBM cells as well as their different invasion patterns. We also review how tumor cells shape their microenvironment and how, vice versa, components of the extracellular matrix and factors from non-neoplastic cells influence tumor cell motility. We further discuss different research platforms for modeling invasion. Finally, we highlight the importance of accounting for the complex interplay between tumor cell invasion and treatment resistance in glioblastoma when considering new therapeutic approaches.

ATF4 contributes to autophagy and survival in sunitinib treated brain tumor initiating cells (BTICs).

Receptor tyrosine kinase (RTK) pathways are known to play an important role in tumor cell proliferation of glioblastoma (GBM). Cellular determinants of RTK-inhibitor sensitivity are important to optimize and tailor treatment strategies. The stress response gene activating transcription factor 4 (ATF4) is involved in homeostasis and cellular protection. However, little is known about its function in GBM. We found that the ATF4/p-eIF2α pathway is activated in response to Sunitinib in primary tumor initiating progenitor cell cultures (BTICs). Furthermore, lysosome entrapment of RTK-inhibitors (RTK-Is) leads to accumulation of autophagosomes. In case of Sunitinib treated cells, autophagy is additionally increased by ATF4 mediated upregulation of autophagy genes. Inhibition of ATF4 by small interfering RNA (siRNA) reduced autophagy and cell proliferation after Sunitinib treatment in a subset of BTIC cultures. Overall, this study suggests a pro-survival role of the ATF4/p-eIF2α pathway in a cell type and treatment specific manner.

Combined Modulation of Tumor Metabolism by Metformin and Diclofenac in Glioma.

Glioblastoma remains a fatal diagnosis. Previous research has shown that metformin, which is an inhibitor of complex I of the respiratory chain, may inhibit some brain tumor initiating cells (BTICs), albeit at dosages that are too high for clinical use. Here, we explored whether a combined treatment of metformin and diclofenac, which is a non-steroidal anti-inflammatory drug (NSAID) shown to inhibit glycolysis by interfering with lactate efflux, may lead to additive or even synergistic effects on BTICs (BTIC-8, -11, -13 and -18) and tumor cell lines (TCs, U87, and HTZ349). Therefore, we investigated the functional effects, including proliferation and migration, metabolic effects including oxygen consumption and extracellular lactate levels, and effects on the protein level, including signaling pathways. Functional investigation revealed synergistic anti-migratory and anti-proliferative effects of the combined treatment with metformin and diclofenac on BTICs and TCs. Signaling pathways did not sufficiently explain synergistic effects. However, we observed that metformin inhibited cellular oxygen consumption and increased extracellular lactate levels, indicating glycolytic rescue mechanisms. Combined treatment inhibited metformin-induced lactate increase. The combination of metformin and diclofenac may represent a promising new strategy in the treatment of glioblastoma. Combined treatment may reduce the effective doses of the single agents and prevent metabolic rescue mechanisms. Further studies are needed in order to determine possible side effects in humans.

Efficacy of D,L-methadone in the treatment of glioblastoma in vitro.

AIM: Recently, D,L-methadone has been put forward as adjuvant treatment in glioblastoma (GBM). METHODS: We analyzed the µ-opioid receptor expression in a set of GBM cell lines and investigated the efficacy of D,L-methadone alone and in combination with temozolomide (TMZ). Results & conclusion: Expression of the µ-opioid receptor was similar in the tested cell lines. High concentrations of D,L-methadone induced apoptosis in all cell lines and showed treatment interaction with TMZ. However, in lower dosages, reflecting clinically attainable concentrations, D,L-methadone alone showed no efficacy, and induced even higher proliferation in one specific cell line. Also, no interaction with TMZ was observed. These results suggest caution to the premature use of D,L-methadone in the treatment of GBM patients.

Stattic and metformin inhibit brain tumor initiating cells by reducing STAT3-phosphorylation.

Glioblastoma (GBM) is the most common and malignant type of primary brain tumor and associated with a devastating prognosis. Signal transducer and activator of transcription number 3 (STAT3) is an important pathogenic factor in GBM and can be specifically inhibited with Stattic. Metformin inhibits GBM cell proliferation and migration. Evidence from other tumor models suggests that metformin inhibits STAT3, but there is no specific data on brain tumor initiating cells (BTICs).We explored proliferation and migration of 7 BTICs and their differentiated counterparts (TCs) after treatment with Stattic, metformin or the combination thereof. Invasion was measured in situ on organotypic brain slice cultures. Protein expression of phosphorylated and total STAT3, as well as AMPK and mTOR signaling were explored using Western blot. To determine functional relevance of STAT3 inhibition by Stattic and metformin, we performed a stable knock-in of STAT3 in selected BTICs.Inhibition of STAT3 with Stattic reduced proliferation in all BTICs, but only in 4 out of 7 TCs. Migration and invasion were equally inhibited in BTICs and TCs. Treatment with metformin reduced STAT3-phosphorylation in all investigated BTICs and TCs. Combined treatment with Stattic and metformin led to significant additive effects on BTIC proliferation, but not migration or invasion. No additive effects on TCs could be detected. Stable STAT3 knock-in partly attenuated the effects of Stattic and metformin on BTICs.In conclusion, metformin was found to inhibit STAT3-phosphorylation in BTICs and TCs. Combined specific and unspecific inhibition of STAT3 might represent a promising new strategy in the treatment of glioblastoma.

P144, a Transforming Growth Factor beta inhibitor peptide, generates antitumoral effects and modifies SMAD7 and SKI levels in human glioblastoma cell lines.

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor, accounting for 60-70% of all gliomas. Current median patient survival time is 14-16 months after diagnosis. Numerous efforts in therapy have not significantly altered the nearly uniform lethality of this malignancy. The Transforming Growth Factor beta (TGF-ß) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-ß pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-ß inhibitor peptide, was analyzed. P144 decreased proliferation, migration, invasiveness, and tumorigenicity in vitro, whereas apoptosis and anoikis were significantly increased for GBM cell lines. SMAD2 phosphorylation was reduced, together with a downregulation of SKI and an upregulation of SMAD7 at both transcriptional and translational levels. Additionally, P144 was able to impair tumor growth and increase survival in an in vivo flank model. Our findings suggest a potential effect of P144 in vitro and in vivo that is mediated by regulation of transcriptional target genes of the TGF-ß pathway, suggesting a therapeutic potential of P144 for GBM treatment.

Metformin inhibits proliferation and migration of glioblastoma cells independently of TGF-ß2.

To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor ß 2 (TGF-ß2) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-ß2-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/-TGF-ß2 revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-ß2 and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-ß2 mRNA expression (qRT-PCR), TGF-ß2 protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-ß2-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.

Validation Study: Response-Predictive Gene Expression Profiling of Glioma Progenitor Cells In Vitro.

BACKGROUND: In a previous publication we introduced a novel approach to identify genes that hold predictive information about treatment outcome. A linear regression model was fitted by using the least angle regression algorithm (LARS) with the expression profiles of a construction set of 18 glioma progenitor cells enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed predicting therapy-induced impairment of proliferation in vitro. Prediction performance was validated in leave one out cross validation. METHODS: In this study, we used an additional validation set of 18 serum-free short-term treated in vitro cell cultures to test the predictive properties of the signature in an independent cohort. We assessed proliferation rates together with transcriptome-wide expression profiles after Sunitinib treatment of each individual cell culture, following the methods of the previous publication. RESULTS: We confirmed treatment-induced expression changes in our validation set, but our signature failed to predict proliferation inhibition. Neither re-calculation of the combined dataset with all 36 BTIC cultures nor separation of samples into TCGA subclasses did generate a proliferation prediction. CONCLUSION: Although the gene signature published from our construction set exhibited good prediction accuracy in cross validation, we were not able to validate the signature in an independent validation data set. Reasons could be regression to the mean, the moderate numbers of samples, or too low differences in the response to proliferation inhibition in the validation set. At this stage and based on the presented results, we conclude that the signature does not warrant further developmental steps towards clinical application.

Adaptive Immune Response to and Survival Effect of Temozolomide- and Valproic Acid-induced Autophagy in Glioblastoma.

BACKGROUND/AIM: The combination of radiotherapy, temozolomide and valproic acid (VPA) has shown some promise in retrospective analyses of patients with glioblastoma, although their mechanisms of action remain unknown. MATERIALS AND METHODS: We investigated the in vitro and in vivo effects of pretreating glioma cells with temozolomide and VPA as an immunization strategy to boost an adaptive immune response in a syngeneic mouse model. RESULTS: Temozolomide and VPA induced autophagy in GL261 glioma cells, and caused tumor antigen-specific T-cells to become activated effector T-cells. Mice with a pre-existing glioma showed no improvement in clinical outcome when immunized with temozolomide- and VPA-treated glioma cells. CONCLUSION: Although temozolomide and VPA treatment of glioma cells can boost the adaptive immune response, in the context of a vaccine therapy, additional factors are necessary to eradicate the tumor and improve survival.

Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with anti-tumorigenic effects in different tumor entities. For glioma, research has generally focused on diclofenac; however data on other NSAIDs, such as ibuprofen, is limited. Therefore, we performed a comprehensive investigation of the cellular, molecular, and metabolic effects of ibuprofen and diclofenac on human glioblastoma cells. METHODS: Glioma cell lines were treated with ibuprofen or diclofenac to investigate functional effects on proliferation and cell motility. Cell cycle, extracellular lactate levels, lactate dehydrogenase-A (LDH-A) expression and activity, as well as inhibition of the Signal Transducer and Activator of Transcription 3 (STAT-3) signaling pathway, were determined. Specific effects of diclofenac and ibuprofen on STAT-3 were investigated by comparing their effects with those of the specific STAT-3 inhibitor STATTIC. RESULTS: Ibuprofen treatment led to a stronger inhibition of cell growth and migration than treatment with diclofenac. Proliferation was affected by cell cycle arrest at different checkpoints by both agents. In addition, diclofenac, but not ibuprofen, decreased lactate levels in all concentrations used. Both decreased STAT-3 phosphorylation; however, diclofenac led to decreased c-myc expression and subsequent reduction in LDH-A activity, whereas treatment with ibuprofen in higher doses induced c-myc expression and less LDH-A alteration. CONCLUSIONS: This study indicates that both ibuprofen and diclofenac strongly inhibit glioma cells, but the subsequent metabolic responses of both agents are distinct. We postulate that ibuprofen may inhibit tumor cells also by COX- and lactate-independent mechanisms after long-term treatment in physiological dosages, whereas diclofenac mainly acts by inhibition of STAT-3 signaling and downstream modulation of glycolysis.

TGF-ß isoforms in cancer: Immunohistochemical expression and Smad-pathway-activity-analysis in thirteen major tumor types with a critical appraisal of antibody specificity and immunohistochemistry assay validity.

The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-ß isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-ß immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

Response-predictive gene expression profiling of glioma progenitor cells in vitro.

BACKGROUND: High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist. METHODS: In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling. RESULTS: As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro. CONCLUSION: For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.