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Purpose: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment., Methods: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX., Results: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis., Conclusion: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy., (© 2023. The Author(s).)

Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor., Competing Interests: JeH is CEO, WW is CSO, and JA, MB, MD and LW are employees of EPO Berlin-Buch GmbH, Berlin, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alcaniz, Winkler, Dahlmann, Becker, Orthmann, Haybaeck, Krassnig, Skofler, Kratzsch, Kuhn, Jödicke, Linnebacher, Fichtner, Walther and Hoffmann.)

Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients' overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I-IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients' survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

Biliary tract cancer (BTC) represents a malignant tumor of the biliary tract including cholangiocarcinoma (CCA) and the carcinoma of the gallbladder (GBC) with a 5-year survival rate between 5 and 18% due to late diagnosis and rapid disease progression. Chronic inflammation is one of the main risk factors for CCA and GBC in particular. IL-6, as a mediator of inflammation, can act through a membrane-bound receptor alpha-chain (mIL-6R, "IL-6 classic signaling") or via soluble forms (sIL-6R, "IL-6 trans-signaling"). However, little is known about the impact on cellular responses of IL-6 trans-signaling on BTC. We analyzed primary tumors as whole sections and as tissue microarrays, and also searched The Cancer Genome Atlas database. Compared to non-neoplastic, non-inflamed gallbladder tissue, IL-6Rα was downregulated in GBC, and this correlated with the patients' overall survival. Furthermore, different CCA cell lines and compounds for activation (IL-6 and Hyper-IL-6) or inhibition (Tocilizumab and sgp130Fc) of IL-6 classic signaling and trans-signaling were used to determine their effects on cellular processes between the two modes of IL-6 signaling. Inhibition of IL-6 trans-signaling by sgp130Fc reduced CCA cell line viability and apoptosis, whereas migration and proliferation were increased. We conclude that IL-6Rα expression is a good prognostic marker for GBC, and that the blocking of IL-6 trans-signaling and activation of IL-6 classic signaling have tumor promoting activity. These findings warrant the exclusion of patients with GBC or other malignancies associated with bile metabolism from IL-6R inhibitor therapy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termed Mallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation., Methods: We analyzed livers of aged Krt18 -/- mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18 -/- mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis., Results: Our analysis of the genetic profile of Krt18 -/- mice with 26 human hepatoma cell lines and with data sets of >300 patients with HCC, where Krt18 -/- gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype., Conclusions: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Glioblastoma is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the role of the epigenome in glioblastoma disease progression. Here, we present genome-scale maps of DNA methylation in matched primary and recurring glioblastoma tumors, using data from a highly annotated clinical cohort that was selected through a national patient registry. We demonstrate the feasibility of DNA methylation mapping in a large set of routinely collected FFPE samples, and we validate bisulfite sequencing as a multipurpose assay that allowed us to infer a range of different genetic, epigenetic, and transcriptional characteristics of the profiled tumor samples. On the basis of these data, we identified subtle differences between primary and recurring tumors, links between DNA methylation and the tumor microenvironment, and an association of epigenetic tumor heterogeneity with patient survival. In summary, this study establishes an open resource for dissecting DNA methylation heterogeneity in a genetically diverse and heterogeneous cancer, and it demonstrates the feasibility of integrating epigenomics, radiology, and digital pathology for a national cohort, thereby leveraging existing samples and data collected as part of routine clinical practice.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Dysregulation of protein synthesis plays a major role in carcinogenesis, a process regulated at multiple levels, including translation of mRNA into proteins. Ribosome assembly requires correct association of ribosome subunits, which is ensured by eukaryotic translation initiation factors (eIFs). eIFs have become targets in cancer therapy studies, and promising data on eIF6 in various cancer entities have been reported. Therefore, we hypothesised that eIF6 represents a crossroad for pulmonary carcinogenesis. High levels of eIF6 are associated with shorter patient overall survival in adenocarcinoma (ADC), but not in squamous cell carcinoma (SQC) of the lung. We demonstrate significantly higher protein expression of eIF6 in ADC and SQC than in healthy lung tissue based on immunohistochemical data from tissue microarrays (TMAs) and on fresh frozen lung tissue. Depletion of eIF6 in ADC and SQC lung cancer cell lines inhibited cell proliferation and induced apoptosis. Knockdown of eIF6 led to pre-rRNA processing and ribosomal 60S maturation defects. Our data indicate that eIF6 is upregulated in NSCLC, suggesting an important contribution of eIF6 to the development and progression of NSCLC and a potential for new treatment strategies against NSCLC., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Formalin fixation prevents tissue autolysis by crosslinking proteins and changes tissue microstructure and MRI signal characteristics. Previous studies showed high variations in MR relaxation time constants of formalin fixed brain tissue, which has been attributed to the use of different formalin concentrations. Our investigations confirmed the influence of formalin concentration on relaxation times and unexpectedly revealed an influence of vendor specific formalin composition, which has not been investigated so far., Methods: We systematically analyzed relaxation times of human brain tissue fixed with 4% and 10% formalin compared with unfixed condition at 3 Tesla MRI. Furthermore, we assessed relaxation times of nine formalin solutions from different vendors and performed comparisons of their magnetic susceptibility by SQUID (superconducting quantum interference device) magnetometry., Results: Tissue relaxation times decreased approximately twice as fast using 10% than in 4% formalin fixation. The vendor specific composition of the formalin solutions and concentration dependent paramagnetic effects showed a substantial contribution to differences in relaxation times of formalin., Conclusion: Our study demonstrates that differences of the formalin composition have substantial effects on MRI signal characteristics after fixation, which can explain the divergence of reported relaxation times beyond the effect of differences in formalin concentration. Magn Reson Med 79:1111-1115, 2018. © 2017 International Society for Magnetic Resonance in Medicine., (© 2017 International Society for Magnetic Resonance in Medicine.)

Colorectal cancer (CRC) is the third most common cause of cancer related death worldwide. Furthermore, with more than 1.2 million cases registered per year, it constitutes the third most frequent diagnosed cancer entity worldwide. Deregulation of protein synthesis has received considerable attention as a major step in cancer development and progression. Eukaryotic translation initiation factors (eIFs) are involved in the regulation of protein synthesis and are functionally linked to the phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. The identification of factors accounting for colorectal carcinoma (CRC) development is a major gap in the field. Besides the importance of eIF3 subunits and the eIF4 complex, eIF1, eIF5 and eIF6 were found to be altered in primary and metastatic CRC. We observed significant difference in the expression profile between low and high grade CRC. eIF1, eIF5 and eIF6 are involved in translational control in CRC. Our findings also indicate a probable clinical impact when separating them into low and high grade colon and rectum carcinoma. eIF and mTOR expression were analysed on protein and mRNA level in primary low and high grade colon carcinoma (CC) and rectum carcinoma (RC) samples in comparison to non-neoplastic tissue without any disease-related pathology. To assess the therapeutic potential of targeting eIF1, eIF5 and eIF6 siRNA knockdown in HCT116 and HT29 cells was performed. We evaluated the eIF knockdown efficacy on protein and mRNA level and investigated proliferation, apoptosis, invasion, as well as colony forming and polysome associated fractions. These results indicate that eIFs, in particular eIF1, eIF5 and eIF6 play a major role in translational control in colon and rectum cancer., Competing Interests: CONFLICTS OF INTEREST The authors disclose no conflicts of interest.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. The initiation of protein translation is an important rate-limiting step in eukaryotes and is crucial in many viral infections. Eukaryotic translation initiation factors (eIFs) are involved in the initiation step of protein translation and are linked to the phosphatidylinositol-3-kinases PI3K/AKT/mTOR pathway. Therefore we aimed to investigate a potential role of eIFs in HCC. We herein report on the immunohistochemical expression of the various eIF subunits in 235 cases of virus-related human HCC. Additionally, we used immunoblot analysis to investigate the expression of virus-related HCC and non-virus-related HCC in comparison to controls. Mammalian target of rapamycin (or mechanistic target of rapamycin as it is known now (mTOR) and activated mTOR were significantly increased in chronic hepatitis C (HCV)-associated HCC, in HCC without a viral background, in alcoholic liver disease and Wilson disease. pPTEN, phosphatase and tensin homologue (PTEN) and pAKT showed a significant increase in HBV- and HCV-associated HCC, chronic hepatitis B, HCC without a viral background, alcoholic steatohepatitis (ASH) and Wilson disease. Phosphorylated (p)-eIF2α, eIF2α, eiF3B, eIF3D, eIF3J, p-eIF4B, eIF4G and eIF6 were upregulated in HCV-associated HCC. eIF2α, p-eIF4B, eIF5 and various eIF3 subunits were significantly increased in chronic hepatitis B (HBV)-associated HCC. HCC without viral background displayed a significant increase for the eIF subunits p-2α, 3C, 3I, 4E and 4G. We noticed engraved differences in the expression pattern between chronic hepatitis B and C, HBV- and HCV-associated HCC and non-virus-related HCC., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: β-Synuclein (β-Syn) is a member of the highly homologous synuclein protein family. The most prominent family member, α-synuclein (α-Syn), abnormally accumulates in so-called Lewy bodies, one of the major pathological hallmarks of α-synucleinopathies. Notably, parts of the peptide backbone, called the nonamyloid component, are also found in amyloid plaques. However, β-Syn seems to have beneficial effects by reducing α-Syn aggregation, and amyloid antiaggregatory activity has been described., Objective: The aim of the study was to analyze if wild-type β-Syn can counteract functional and pathological changes in a murine Alzheimer model over different time periods., Methods: At the onset of pathology, lentiviral particles expressing human β-Syn were injected into the hippocampus of transgenic mice overexpressing human amyloid precursor protein with Swedish and London mutations (APPSL). An empty vector served as the control. Behavioral analyses were performed 1, 3 and 6 months after injection followed by biochemical and histological examinations of brain samples., Results: β-Syn expression was locally concentrated and rather modest, but nevertheless changed its effect on APP expression and plaque load in a time- and concentration-dependent manner. Interestingly, the phosphorylation of glycogen synthase kinase 3 beta was enhanced in APPSL mice expressing human β-Syn, but an inverse trend was observed in wild-type animals., Conclusion: The initially reported beneficial effects of β-Syn could be partially reproduced, but locally elevated levels of β-Syn might also cause neurodegeneration. To enlighten the controversial pathological mechanism of β-Syn, further examinations considering the relationship between concentration and exposure time of β-Syn are needed., (© 2015 S. Karger AG, Basel.)

Structuring of thin polymer layers by soft lithography with template bioparticles results in the formation of selective surface cavities, leading to highly effective sensor systems when combined with mass-sensitive transducers, especially QCM. These sensors allow selective differentiation of various stages of development of yeast cells. In order to achieve a higher degree of standardisation, we fabricated plastic yeast cells and utilised them for the stamp imprinting procedures. These sensitive layers are capable of the differentiation between Saccharomyces cerevisiae and Saccharomyces bayanus. Aside from achieving the same sensitivity compared to the polymers that were structured using native cells, we realised further enhancement of selectivity exceeding a factor of three regarding the two cell strains. These ideas could also be transferred to develop a recognition system for the more flexible erythrocytes and therefore MIP-layers of polyvinylpyrrolidone were combined with QCMs. These devices provide sensor-based ABO blood group typing. Additionally, the differentiation of the subgroups A(1) and A(2) is shown with the generated MIP-layers that are decorated by high selectivity, namely the threefold frequency effect for the imprinted template, and negligible unspecific effects. Application of soft lithographic methods furthermore allows the design of artificial erythrocytes. These "plastic" blood cells possess an increased robustness compared to the native cells, thus opening up multiple novel strategies of surface patterning.

Self-organized receptor layers are synthesized by molecular imprinting methods directly on pre-coated 10 MHz quartz-crystal microbalances (QCMs). The surface-imprinting is performed by three methods using amorphous, crystalline and solubilized trypsin, respectively, as templates. These attempts allowed us to compare imprinting results obtained with templating proteins in the dry state as well as in aqueous solution. All methods are generally applicable for surface imprinting of thin films. The biomimetic sensor layers allow selective enzyme enrichment on the imprinted electrode with detection limits as low as 100 ng ml(-1) and response times of a few minutes. Solution-based polymer imprinting with native trypsin as template resulted in the highest specific enzyme recognition, which even allowed us to distinguish denatured trypsin from the native form.