Arabel Vollmann-Zwerenz

Cell type specific applicability of 5-ethynyl-2′-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry

Using the nucleoside analogue EdU (5‐ethynyl‐2′‐deoxyuridine) for thymidine substitution instead of BrdU (5‐bromo‐2′‐deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK‐BR‐3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK‐BR‐3 breast cancer cells was highly affected by long term exposure to EdU. Both SK‐BR‐3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK‐BR‐3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

Humanized tumor mice—A new model to study and manipulate the immune response in advanced cancer therapy

The immunological impact on antibody‐based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD‐scid IL2Rγnull mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor‐cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T‐cell maturation and tumor cell‐specific T‐cell activation. In addition, Natural–Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL‐15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody‐dependent cellular cytotoxicity‐based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system‐related target cancer therapy and resistance.

Lactate-Modulated Induction of THBS-1 Activates Transforming Growth Factor (TGF)-beta2 and Migration of Glioma Cells In Vitro

Background An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein. Methods Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays. Results Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells. Conclusion We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

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.

Multichromatic phenotyping of HER receptor coexpression in breast tumor tissue samples using flow cytometry--possibilities and limitations.

The prognostic significance of HER2 expression in human breast carcinomas is beyond dispute nowadays. The HER family of receptor tyrosine kinases comprises four members (HER1/ErbB1/EGFR, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) that act in concert via transactivation and consequently compose a functional signaling unit. Besides HER2 overexpression, coexpression of other HER receptors has substantial impact on course of disease and potential therapeutic benefit. This observation is substantiated by numerous preclinical studies and retrospective studies done on patients with breast cancer. Against this background, the quantification of all HER receptor expressions at the same time would significantly extend the information content revealed by routine diagnosis of breast cancer tissues. Moreover, the knowledge of HER receptor coexpression profiles in primary tumor samples could provide the basis to design and develop highly specific antireceptor treatment strategies. Here, we report on a simultaneous flow cytometric detection of all four HER receptors on carcinoma cells isolated from primary breast cancer tissues and separated from nonepithelial cells by cytokeratin staining. Combined with DNA, i.e. ploidy quantification, the approach resulted in a six‐parameter assay that could complement the diagnosis of a variety of diseases in which HER receptor expression has a pivotal impact on the degree of malignancy.

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.

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-Δ in cancer including data on the expression or activation of TGF-Δ pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-Δ pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-Δ 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-Δ1, TGF-Δ2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-Δ isoform directed therapies. Of note, TGF-Δ antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-β 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-Δ directed antisense therapies based upon TGF-β immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

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-β) signaling pathway plays a key role in GBM and is implicated in proliferation, invasion and therapy resistance. Several inhibitors of the TGF-β pathway have entered clinical trials or are under development. In this work, the therapeutic potential of P144, a TGF-β 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-β pathway, suggesting a therapeutic potential of P144 for GBM treatment.

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.

Versican isoform V1 regulates proliferation and migration in high-grade gliomas

Versican is a large chondroitin sulphate proteoglycan produced by several tumor cell types, including high-grade gliomas. Increased expression of distinct versican isoforms in the extracellular matrix plays a role in tumor cell growth, adhesion and migration. We have recently shown that transforming growth factor (TGF-beta)2, an important modulator of glioma invasion, interacts with versican isoforms V0/V1 during malignant progression of glioma in vitro. However, the distinct subtype of versican that modulates these effects could not be specified. Here, we show that transient down-regulation of V1 by siRNA leads to a significant reduction of proliferation and migration in glioblastoma cell lines and glioblastoma progenitor cells, whereas tumor cell attachment stays unaffected. We conclude that V1 plays a predominant role in modulating central pathophysiological mechanisms as proliferation and migration in glioblastoma. Considering that TGF-beta is a master regulator of glioma pathophysiology, and that V0/1 is induced by TGF-beta2, therapeutic regulation of V1 may induce meaningful effects on glioma cell migration not only in vitro, but also in vivo.