Tryptophan metabolism is inversely regulated in the tumor and blood of patients with glioblastoma.

Tryptophan (Trp)-catabolic enzymes (TCEs) produce metabolites that activate the aryl hydrocarbon receptor (AHR) and promote tumor progression and immunosuppression in glioblastoma. As therapies targeting TCEs or AHR become available, a better understanding of Trp metabolism is required. Methods: The combination of LC-MS/MS with chemical isobaric labeling enabled the simultaneous quantitative comparison of Trp and its amino group-bearing metabolites in multiple samples. We applied this method to the sera of a cohort of 43 recurrent glioblastoma patients and 43 age- and sex-matched healthy controls. Tumor volumes were measured in MRI data using an artificial neural network-based approach. MALDI MSI visualized Trp and its direct metabolite N-formylkynurenine (FK) in glioblastoma tissue. Analysis of scRNA-seq data was used to detect the presence of Trp metabolism and AHR activity in different cell types in glioblastoma. Results: Compared to healthy controls, glioblastoma patients showed decreased serum Trp levels. Surprisingly, the levels of Trp metabolites were also reduced. The decrease became smaller with more enzymatic steps between Trp and its metabolites, suggesting that Trp availability controls the levels of its systemic metabolites. High tumor volume associated with low systemic metabolite levels and low systemic kynurenine levels associated with worse overall survival. MALDI MSI demonstrated heterogeneity of Trp catabolism across glioblastoma tissues. Analysis of scRNA-seq data revealed that genes involved in Trp metabolism were expressed in almost all the cell types in glioblastoma and that most cell types, in particular macrophages and T cells, exhibited AHR activation. Moreover, high AHR activity associated with reduced overall survival in the glioblastoma TCGA dataset. Conclusion: The novel techniques we developed could support the identification of patients that may benefit from therapies targeting TCEs or AHR activation.

Direct Automated MALDI Mass Spectrometry Analysis of Cellular Transporter Function: Inhibition of OATP2B1 Uptake by 294 Drugs.

OATP2B1, a member of the solute carrier (SLC) transporter family, is an important mechanism of substrate drug uptake in the intestine and liver and therefore a determinant of clinical pharmacokinetics and site of drug-drug interactions. Other SLC transporters have emerged as pharmacology targets. Studies of SLC transporter uptake to-date relied on radioisotope- or fluorescence-labeled reagents or low-throughput quantification of unlabeled compounds in cell lysate. In this study, we developed a cell-based MALDI MS workflow for investigation of OATP2B1 cellular uptake by optimizing the substrate, matrix, matrix-analyte ratio, and matrix application and normalization method. This workflow was automated and applied to characterize substrate transport kinetics and to test 294 top-marketed drugs for OATP2B1 inhibition and quantify inhibitory potencies necessary for extrapolation of clinical drug-drug interaction potential. Intra-assay reproducibility of this MALDI MS method was high (CV < 10%), and results agreed well (83% overlap) with previously published radioisotope assay data. Our results indicate that fast and robust MALDI MS cellular assays could emerge as a high-throughput label-free alternative for direct assessment of drug transporter function in DDIs and toxicities as well as enable drug discovery for transporters as pharmacology targets.

Quantitative Mass Spectrometry Imaging Reveals Mutation Status-independent Lack of Imatinib in Liver Metastases of Gastrointestinal Stromal Tumors.

Mass spectrometry imaging (MSI) is an enabling technology for label-free drug disposition studies at high spatial resolution in life science- and pharmaceutical research. We present the first extensive clinical matrix-assisted laser desorption/ionization (MALDI) quantitative mass spectrometry imaging (qMSI) study of drug uptake and distribution in clinical specimen, analyzing 56 specimens of tumor and corresponding non-tumor tissues from 27 imatinib-treated patients with the biopsy-proven rare disease gastrointestinal stromal tumors (GIST). For validation, we compared MALDI-TOF-qMSI with conventional UPLC-ESI-QTOF-MS-based quantification from tissue extracts and with ultra-high resolution MALDI-FTICR-qMSI. We introduced a novel generalized nonlinear calibration model of drug quantities based on computational evaluation of drug-containing areas that enabled better data fitting and assessment of the inherent method nonlinearities. Imatinib tissue spatial maps revealed striking inefficiency in drug penetration into GIST liver metastases even though the corresponding healthy liver tissues in the vicinity showed abundant imatinib levels beyond the limit of quantification (LOQ), thus providing evidence for secondary drug resistance independent of mutation status. Taken together, these findings underscore the important application of MALDI-qMSI in studying the spatial distribution of molecularly targeted therapeutics in oncology, namely to serve as orthogonal post-surgical approach to evaluate the contribution of anticancer drug disposition to resistance against treatment.

Analyzing the antiseptic capacity of silver-functionalized poly(ethylene glycol)-heparin hydrogels after human whole blood exposure.

Advanced blood contacting biomaterials are designed to combine antiseptic and anticoagulant functionalities. Here, we present a new in vitro methodology for the analysis of bacterial adhesion and growth after the preceding human whole blood incubation of the tested materials. Poly(styrene) surfaces as well as thrombin-responsive and non-responsive poly(ethylene glycol)-heparin hydrogel coatings, with and without silver functionalization, were analyzed with this approach using freshly drawn human whole blood and various human pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). Adsorbed blood proteins and adherent immune cells were observed to suppress bacterial colonization on poly(styrene) surfaces. Silver functionalization of responsive and non-responsive poly(ethylene glycol)-heparin hydrogels had no influence on microbial attachment but decreased bacterial proliferation and viability. Whole blood pre-incubation did not affect the antimicrobial properties of the tested silver-modified hydrogels. In sum, our introduced multistage incubation test revealed the antibacterial effects as well as antiseptic-permissive characteristics of blood-borne interfacial layers on polymeric biomaterials.

Fourier Transform Infrared Microscopy Enables Guidance of Automated Mass Spectrometry Imaging to Predefined Tissue Morphologies.

Multimodal imaging combines complementary platforms for spatially resolved tissue analysis that are poised for application in life science and personalized medicine. Unlike established clinical in vivo multimodality imaging, automated workflows for in-depth multimodal molecular ex vivo tissue analysis that combine the speed and ease of spectroscopic imaging with molecular details provided by mass spectrometry imaging (MSI) are lagging behind. Here, we present an integrated approach that utilizes non-destructive Fourier transform infrared (FTIR) microscopy and matrix assisted laser desorption/ionization (MALDI) MSI for analysing single-slide tissue specimen. We show that FTIR microscopy can automatically guide high-resolution MSI data acquisition and interpretation without requiring prior histopathological tissue annotation, thus circumventing potential human-annotation-bias while achieving >90% reductions of data load and acquisition time. We apply FTIR imaging as an upstream modality to improve accuracy of tissue-morphology detection and to retrieve diagnostic molecular signatures in an automated, unbiased and spatially aware manner. We show the general applicability of multimodal FTIR-guided MALDI-MSI by demonstrating precise tumor localization in mouse brain bearing glioma xenografts and in human primary gastrointestinal stromal tumors. Finally, the presented multimodal tissue analysis method allows for morphology-sensitive lipid signature retrieval from brains of mice suffering from lipidosis caused by Niemann-Pick type C disease.

Inhibition of Rho-Associated Kinase 1/2 Attenuates Tumor Growth in Murine Gastric Cancer.

Gastric cancer (GC) remains a malignant disease with high mortality. Patients are frequently diagnosed in advanced stages where survival prognosis is poor. Thus, there is high medical need to find novel drug targets and treatment strategies. Recently, the comprehensive molecular characterization of GC subtypes revealed mutations in the small GTPase RHOA as a hallmark of diffuse-type GC. RHOA activates RHO-associated protein kinases (ROCK1/2) which regulate cell contractility, migration and growth and thus may play a role in cancer. However, therapeutic benefit of RHO-pathway inhibition in GC has not been shown so far. The ROCK1/2 inhibitor 1-(5-isoquinoline sulfonyl)-homopiperazine (HA-1077, fasudil) is approved for cerebrovascular bleeding in patients. We therefore investigated whether fasudil (i.p., 10 mg/kg per day, 4 times per week, 4 weeks) inhibits tumor growth in a preclinical model of GC. Fasudil evoked cell death in human GC cells and reduced the tumor size in the stomach of CEA424-SV40 TAg transgenic mice. Small animal PET/CT confirmed preclinical efficacy. Mass spectrometry imaging identified a translatable biomarker for mouse GC and suggested rapid but incomplete in situ distribution of the drug to gastric tumor tissue. RHOA expression was increased in the neoplastic murine stomach compared with normal non-malignant gastric tissue, and fasudil reduced (auto) phosphorylation of ROCK2 at THR249 in vivo and in human GC cells in vitro. In sum, our data suggest that RHO-pathway inhibition may constitute a novel strategy for treatment of GC and that enhanced distribution of future ROCK inhibitors into tumor tissue may further improve efficacy.

The stress kinase GCN2 does not mediate suppression of antitumor T cell responses by tryptophan catabolism in experimental melanomas.

Tryptophan metabolism is a key process that shapes the immunosuppressive tumor microenvironment. The two rate-limiting enzymes that mediate tryptophan depletion, indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO), have moved into the focus of research and inhibitors targeting IDO and TDO have entered clinical trials. Local tryptophan depletion is generally viewed as the crucial immunosuppressive mechanism. In T cells, the kinase general control non-derepressible 2 (GCN2) has been identified as a molecular sensor of tryptophan deprivation. GCN2 activation by tryptophan depletion induces apoptosis and mitigates T cell proliferation. Here, we investigated whether GCN2 attenuates tumor rejection in experimental B16 melanoma using T cell-specific Gcn2 knockout mice. Our data demonstrate that GCN2 in T cells did not affect immunity to B16 tumors even when animals were treated with antibodies targeting cytotoxic T lymphocyte antigen-4 (CTLA4). GCN2-deficient gp100 TCR-transgenic T cells were equally effective as wild-type pmel T cells against gp100-expressing B16 melanomas after adoptive transfer and gp100 peptide vaccination. Even augmentation of tumoral tryptophan metabolism in B16 tumors by lentiviral overexpression of Tdo did not differentially affect GCN2-proficient vs. GCN2-deficient T cells in vivo. Importantly, GCN2 target genes were not upregulated in tumor-infiltrating T cells. MALDI-TOF MS imaging of B16 melanomas demonstrated maintenance of intratumoral tryptophan levels despite high tryptophan turnover, which prohibits a drop in tryptophan sufficient to activate GCN2 in tumor-infiltrating T cells. In conclusion, our results do not suggest that suppression of antitumor immune responses by tryptophan metabolism is driven by local tryptophan depletion and subsequent GCN2-mediated T cell anergy.

Clinical Presentation and Multi-Parametric Screening Surrogates of Ischemic Stroke Patients Suffering from Infective Endocarditis.

BACKGROUND: Infective endocarditis (IE) represents a life-threatening condition due to complications like cardiac failure and thromboembolism. In ischemic stroke, IE formally excludes patients from approaches addressing the recanalization of occluded vessels, challenging decision-making in the early phase of hospitalization. This study aimed at the rate and clinical course of stroke patients with IE and explored clinical, imaging-based and serum parameters, which would allow early identification. METHODS: A hospital-based registry containing 1,531 ischemic stroke patients was screened for IE identified by echocardiography. In addition to clinical parameters, patterns of cerebral manifestation as well as a variety of inflammatory serum and myocardial markers were analyzed concerning their predictive impact for identifying affected patients. RESULTS: IE was found in 26 patients (1.7%) and was associated with an increased body temperature and cardiac murmurs. Patients suffering from IE demonstrated a more severe clinical affection at hospital discharge and an impaired symptom decline during hospitalization, further deteriorated by the use of systemic thrombolysis. Distribution of cerebral infarction patterns did not differ between the groups. C-reactive protein (CRP) and leukocyte count as well as troponin and myoglobin, taken at hospital admission, were found to be significantly associated with IE. CONCLUSIONS: IE in stroke patients is associated with worse clinical outcome, complicated by intravenously applied thrombolysis, and therefore needs to be screened during the early phase of hospitalization. Increased serum levels of CRP and leukocyte count in combination with an increased body temperature or abnormal cardiac murmurs should entail rapid initiation of further diagnostics, that is, transoesophageal echocardiography.

DMSO-enhanced MALDI MS imaging with normalization against a deuterated standard for relative quantification of dasatinib in serial mouse pharmacology studies.

Matrix-assisted laser desorption/ionization mass spectrometry imaging is an emerging powerful technique in drug metabolism and pharmacokinetics research. Despite recent progress in mass-spectrometry-based localization and relative quantification of small-molecule drugs and their metabolites in tissue, improved methods for drug extraction/ionization are required. Furthermore, relative quantification of drugs by mass spectrometry imaging in larger rodent cohorts is a necessary proof-of-concept study to demonstrate the utility of such a workflow in an industrial setting. Using as an example the tyrosine kinase inhibitor dasatinib, a leukemia drug, we demonstrate that inclusion of dimethyl sulfoxide in standard matrix solutions significantly improves ion intensity in mass spectrometry images and reveals enrichment of the drug in mouse kidney medulla. We furthermore show in a time-course study in multiple mice that normalization against a deuterated internal standard, dasatinib-D8, which is applied together with the matrix, makes possible relative quantification of the drug that correlates well with canonical liquid chromatography­tandem mass spectrometry based drug quantification.

Comparing endoscopic systems on two simulated tasks.

Endoscopes are slender instruments for performing medical procedures through small incisions or natural body orifices. Three experiments examined the performance effects of various endoscope systems when naive participants executed tasks in a bladder-like environment. The systems involved a direct endoscope, requiring the user to look through a lens at the outer part of the endoscope, and a video endoscope that picks up the image via a camera attached to the endoscope and displays it on a video monitor. The results indicate that the participants performed the tasks better with video than with direct endoscopes, and showed more transfer of practice with video endoscopic surgery from a small to a large task environment than vice versa. The optimal position of the monitor with video endoscopic surgery appeared to involve a reasonable angle relative to the operating area (45 degrees was used). Performance was less at a greater angle (90 degrees), but also at a small angle (10 degrees). It did not matter whether the monitor was located left, right or above the operating area. As performance did not fully transfer to differently sized task environments, endoscopists should initially train with task environments of different, and especially small, sizes.

Distribution of new human beta-defensin genes clustered on chromosome 20 in functionally different segments of epididymis.

Human beta-defensins are a family of cationic peptides that share a pattern of six conserved cysteine residues. We describe the cloning and characterization of the cDNAs of five novel beta-defensin genes (DEFB25-DEFB29) clustered on chromosome 20p13, which were identified using a bioinformatics approach. Expression analysis revealed the occurrence of the transcripts in only a few organs, with the highest abundance in the male genital tract. Examination of beta-defensin expression in human epididymis by real-time quantitative RT-PCR revealed a distribution along the functionally different segments of the epididymal duct. In situ hybridization for one of the cDNAs shows mRNA restriction to the epithelial cell layer of the epididymis, known to secrete factors responsible for sperm maturation. We suggest that the novel peptides carry out physiological functions in the male genital tract that may not be directly related to bacterial growth inhibition in host defense.