Primary and hTERT-Transduced Mesothelioma-Associated Fibroblasts but Not Primary or hTERT-Transduced Mesothelial Cells Stimulate Growth of Human Mesothelioma Cells.

Pleural mesothelioma (PM) is an aggressive malignancy that develops in a unique tumor microenvironment (TME). However, cell models for studying the TME in PM are still limited. Here, we have generated and characterized novel human telomerase reverse transcriptase (hTERT)-transduced mesothelial cell and mesothelioma-associated fibroblast (Meso-CAF) models and investigated their impact on PM cell growth. Pleural mesothelial cells and Meso-CAFs were isolated from tissue of pneumothorax and PM patients, respectively. Stable expression of hTERT was induced by retroviral transduction. Primary and hTERT-transduced cells were compared with respect to doubling times, hTERT expression and activity levels, telomere lengths, proteomes, and the impact of conditioned media (CM) on PM cell growth. All transduced derivatives exhibited elevated hTERT expression and activity, and increased mean telomere lengths. Cell morphology remained unchanged, and the proteomes were similar to the corresponding primary cells. Of note, the CM of primary and hTERT-transduced Meso-CAFs stimulated PM cell growth to the same extent, while CM derived from mesothelial cells had no stimulating effect, irrespective of hTERT expression. In conclusion, all new hTERT-transduced cell models closely resemble their primary counterparts and, hence, represent valuable tools to investigate cellular interactions within the TME of PM.

Multiomics-empowered Deep Phenotyping of Ulcerative Colitis Identifies Biomarker Signatures Reporting Functional Remission States.

INTRODUCTION: Ulcerative colitis [UC] is a chronic disease with rising incidence and unclear aetiology. Deep molecular phenotyping by multiomics analyses may provide novel insights into disease processes and characteristic features of remission states. METHODS: UC pathomechanisms were assessed by proteome profiling of human tissue specimens, obtained from five distinct colon locations for each of the 12 patients included in the study. Systemic disease-associated alterations were evaluated thanks to a cross-sectional setting of mass spectrometry-based multiomics analyses comprising proteins, metabolites, and eicosanoids of plasma obtained from UC patients during acute episodes and upon remission, in comparison with healthy controls. RESULTS: Tissue proteome profiling indicated colitis-associated activation of neutrophils, macrophages, B and T cells, fibroblasts, endothelial cells and platelets, and hypoxic stress, and suggested a general downregulation of mitochondrial proteins accompanying the establishment of apparent wound healing-promoting activities including scar formation. Whereas pro-inflammatory proteins were apparently upregulated by immune cells, the colitis-associated epithelial cells, fibroblasts, endothelial cells, and platelets seemed to predominantly contribute anti-inflammatory and wound healing-promoting proteins. Blood plasma proteomics indicated chronic inflammation and platelet activation, whereas plasma metabolomics identified disease-associated deregulations of gut and gut microbiome-derived metabolites. Upon remission several, but not all, molecular candidate biomarker levels recovered back to normal. CONCLUSION: The findings may indicate that microvascular damage and platelet deregulation hardly resolve upon remission, but apparently persist as disease-associated molecular signatures. This study presents local and systemic molecular alterations integrated in a model for UC pathomechanisms, potentially supporting the assessment of disease and remission states in UC patients.

Mesothelioma-associated fibroblasts enhance proliferation and migration of pleural mesothelioma cells via c-Met/PI3K and WNT signaling but do not protect against cisplatin.

BACKGROUND: Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells. METHODS: Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. RESULTS: Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib. CONCLUSIONS: Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.

Finger sweat analysis enables short interval metabolic biomonitoring in humans.

Metabolic biomonitoring in humans is typically based on the sampling of blood, plasma or urine. Although established in the clinical routine, these sampling procedures are often associated with a variety of compliance issues, which are impeding time-course studies. Here, we show that the metabolic profiling of the minute amounts of sweat sampled from fingertips addresses this challenge. Sweat sampling from fingertips is non-invasive, robust and can be accomplished repeatedly by untrained personnel. The sweat matrix represents a rich source for metabolic phenotyping. We confirm the feasibility of short interval sampling of sweat from the fingertips in time-course studies involving the consumption of coffee or the ingestion of a caffeine capsule after a fasting interval, in which we successfully monitor all known caffeine metabolites as well as endogenous metabolic responses. Fluctuations in the rate of sweat production are accounted for by mathematical modelling to reveal individual rates of caffeine uptake, metabolism and clearance. To conclude, metabotyping using sweat from fingertips combined with mathematical network modelling shows promise for broad applications in precision medicine by enabling the assessment of dynamic metabolic patterns, which may overcome the limitations of purely compositional biomarkers.

EGF Induces Migration Independent of EMT or Invasion in A549 Lung Adenocarcinoma Cells.

Tumors and the tumor microenvironment produce multiple growth factors that influence cancer cell behavior via various signal transduction pathways. Growth factors, like transforming growth factor ß (TGFß) and epidermal growth factor (EGF), have been shown to induce proliferation, migration, and invasion in different cell models. Both factors are frequently overexpressed in cancer and will often act in combination. Although both factors are being used as rational targets in clinical oncology, the similarities and differences of their contributions to cancer cell migration and invasion are not fully understood. Here we compared the impact of treating A549 lung adenocarcinoma cells with TGFß, EGF, and both in combination by applying videomicroscopy, functional assays, immunoblotting, real-time PCR, and proteomics. Treatment with both factors stimulated A549 migration to a similar extent, but with different kinetics. The combination had an additive effect. EGF-induced migration depended on activation of the mitogen-activated protein kinase (MAPK) pathway. However, this pathway was dispensable for TGFß-induced migration, despite a strong activation of this pathway by TGFß. Proteome analysis (data are available via ProteomeXchange with identifier PXD023024) revealed an overlap in expression patterns of migration-related proteins and associated gene ontology (GO) terms by TGFß and EGF. Further, only TGFß induced the expression of epithelial to mesenchymal transition (EMT)-related proteins like matrix metalloproteinase 2 (MMP2). EGF, in contrast, made no major contribution to EMT marker expression on either the protein or the transcript level. In line with these expression patterns, TGFß treatment significantly increased the invasive capacity of A549 cells, while EGF treatment did not. Moreover, the addition of EGF failed to enhance TGFß-induced invasion. Overall, these data suggest that TGFß and EGF can partly compensate for each other for stimulation of cell migration, but abrogation of TGFß signaling may be more suitable to suppress cell invasion.

Membrane disruption, but not metabolic rewiring, is the key mechanism of anticancer-action of FASN-inhibitors: a multi-omics analysis in ovarian cancer.

Fatty-acid(FA)-synthase(FASN) is a druggable lipogenic oncoprotein whose blockade causes metabolic disruption. Whether drug-induced metabolic perturbation is essential for anticancer drug-action, or is just a secondary-maybe even a defence response-is still unclear. To address this, SKOV3 and OVCAR3 ovarian cancer(OC) cell lines with clear cell and serous histology, two main OC subtypes, were exposed to FASN-inhibitor G28UCM. Growth-inhibition was compared with treatment-induced cell-metabolomes, lipidomes, proteomes and kinomes. SKOV3 and OVCAR3 were equally sensitive to low-dose G28UCM, but SKOV3 was more resistant than OVCAR3 to higher concentrations. Metabolite levels generally decreased upon treatment, but individual acylcarnitines, glycerophospholipids, sphingolipids, amino-acids, biogenic amines, and monosaccharides reacted differently. Drug-induced effects on central-carbon-metabolism and oxidative-phosphorylation (OXPHOS) were essentially different in the two cell lines, since drug-naïve SKOV3 are known to prefer glycolysis, while OVCAR3 favour OXPHOS. Moreover, drug-dependent increase of desaturases and polyunsaturated-fatty-acids (PUFAs) were more pronounced in SKOV3 and appear to correlate with G28UCM-tolerance. In contrast, expression and phosphorylation of proteins that control apoptosis, FA synthesis and membrane-related processes (beta-oxidation, membrane-maintenance, transport, translation, signalling and stress-response) were concordantly affected. Overall, membrane-disruption and second-messenger-silencing were crucial for anticancer drug-action, while metabolic-rewiring was only secondary and may support high-dose-FASN-inhibitor-tolerance. These findings may guide future anti-metabolic cancer intervention.

Neutrophil Extracellular Trap Formation Correlates with Favorable Overall Survival in High Grade Ovarian Cancer.

It is still a question of debate whether neutrophils, often found in the tumor microenvironment, mediate tumor-promoting or rather tumor-inhibiting activities. The present study focuses on the involvement of neutrophils in high grade serous ovarian cancer (HGSOC). Macroscopic features classify two types of peritoneal tumor spread in HGSOC. Widespread and millet sized lesions characterize the miliary type, while non-miliary metastases are larger and associated with better prognosis. Multi-omics and FACS data were generated from ascites samples. Integrated data analysis demonstrates a significant increase of neutrophil extracellular trap (NET)-associated molecules in non-miliary ascites samples. A co-association network analysis performed with the ascites data further revealed a striking correlation between NETosis-associated metabolites and several eicosanoids. The congruence of data generated from primary neutrophils with ascites analyses indicates the predominance of NADPH oxidase 2 (NOX)-independent NETosis. NETosis is associated with protein S100A8/A9 release. An increase of the S100A8/CRP abundance ratio was found to correlate with favorable survival of HGSOC patients. The analysis of additional five independent proteome studies with regard to S100A8/CRP ratios confirmed this observation. In conclusion, NET formation seems to relate with better cancer patient outcome.

Proteome Analysis Reveals Distinct Mitochondrial Functions Linked to Interferon Response Patterns in Activated CD4+ and CD8+ T Cells.

While genetic traits and epigenetic modifications mainly encode cell type-specific effector functions, the eventual outcome is also prone to modulation by post-transcriptional regulation mechanisms. T cells are a powerful model for the investigation of such modulatory effects, as common precursor cells may differentiate either to helper CD4+ T cells or cytotoxic CD8+ cells, which elicit distinct functionalities upon TCR-stimulation. Human primary CD4+ and CD8+ T cells were purified from three individual donors and activated with anti-CD3/CD28 antibodies. Associated proteome alterations were analyzed by high-resolution mass spectrometry using a label-free shotgun approach. Metabolic activation was indicated by upregulation of enzymes related to glycolysis, NADH production, fatty acid synthesis, and uptake as well as amino acid and iron uptake. Besides various inflammatory effector molecules, the mitochondrial proteins CLUH, TFAM, and TOMM34 were found specifically induced in CD4+ T cells. Investigation of overrepresented conserved transcription binding sites by the oPOSSUM software suggested interferon type I inducer IRF1 to cause many of the observed proteome alterations in CD4+ T cells. RT qPCR demonstrated the specific induction of IRF1 in CD4+ T cells only. While the interferon regulatory factor IRF4 was found induced in both T cell subtypes at protein and mRNA level, IRF9 and the type I interferon-induced proteins IFIT1, IFIT3, and MX1 were only found induced in CD4+ T cells. As oxidative stress enhances mitochondrial DNA-dependent type I interferon responses, the present data suggested that mitochondrial activities regulate those cell type-specific signaling pathways. Indeed, we detected mitochondrial superoxide formation predominantly in CD4+ T cells via FACS analysis with MitoSOX™ and confirmed this observation by live cell imaging with confocal microscopy. As interferon signaling regulates important features such as resistance regarding immune checkpoint blockade therapy, the present data may identify potential new targets for the efficient control of highly relevant immune cell properties.

Metabolic, Anti-apoptotic and Immune Evasion Strategies of Primary Human Myeloma Cells Indicate Adaptations to Hypoxia.

Multiple Myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). It develops from a premalignant stage, monoclonal gammopathy of undetermined significance (MGUS), often via an intermediate stage, smoldering MM (SMM). The mechanisms of MM progression have not yet been fully understood, all the more because patients with MGUS and SMM already carry similar initial mutations as found in MM cells. Over the last years, increased importance has been attributed to the tumor microenvironment and its role in the pathophysiology of the disease. Adaptations of MM cells to hypoxic conditions in the BM have been shown to contribute significantly to MM progression, independently from the genetic predispositions of the tumor cells. Searching for consequences of hypoxia-induced adaptations in primary human MM cells, CD138-positive plasma cells freshly isolated from BM of patients with different disease stages, comprising MGUS, SMM, and MM, were analyzed by proteome profiling, which resulted in the identification of 6218 proteins. Results have been made fully accessible via ProteomeXchange with identifier PXD010600. Data previously obtained from normal primary B cells were included for comparative purposes. A principle component analysis revealed three clusters, differentiating B cells as well as MM cells corresponding to less and more advanced disease stages. Comparing these three clusters pointed to the alteration of pathways indicating adaptations to hypoxic stress in MM cells on disease progression. Protein regulations indicating immune evasion strategies of MM cells were determined, supported by immunohistochemical staining, as well as transcription factors involved in MM development and progression. Protein regulatory networks related to metabolic adaptations of the cells became apparent. Results were strengthened by targeted analyses of a selected panel of metabolites in MM cells and MM-associated fibroblasts. Based on our data, new opportunities may arise for developing therapeutic strategies targeting myeloma disease progression.

Curcumin exerts its antitumor effects in a context dependent fashion.

Proteome profiling profoundly contributes to the understanding of cell response mechanisms to drug actions. Such knowledge may become a key to improve personalized medicine. In the present study, the effects of the natural remedy curcumin on breast cancer model systems were investigated. MCF-7, ZR-75-1 and TGF-ß1 pretreated fibroblasts, mimicking cancer-associated fibroblasts (CAFs), were treated independently as well as in tumor cell/CAF co-cultures. Remarkably, co-culturing with CAF-like cells (CLCs) induced different proteome alterations in MCF-7 and ZR-75-1 cells, respectively. Curcumin significantly induced HMOX1 in single cell type models and co-cultures. However, other curcumin effects differed. In the MCF-7/CLC co-culture, curcumin significantly down-regulated RC3H1, a repressor of inflammatory signaling. In the ZR-75-1/CLC co-culture, curcumin significantly down-regulated PEG10, an anti-apoptotic protein, and induced RRAGA, a pro-apoptotic protein involved in TNF-alpha signaling. Furthermore, curcumin induced AKR1C2, an important enzyme for progesterone metabolism. None of these specific curcumin effects were observed in single cell type cultures. All high-resolution mass spectrometry data are available via ProteomeXchange with the identifier PXD008719. The present data demonstrate that curcumin induces proteome alterations, potentially accounting for its known antitumor effects, in a strongly context-dependent fashion. BIOLOGICAL SIGNIFICANCE: Better means to understand and potentially predict individual variations of drug effects are urgently required. The present proteome profiling study of curcumin effects demonstrates the massive impact of the cell microenvironment on cell responses to drug action. Co-culture models apparently provide more biologically relevant information regarding curcumin effects than single cell type cultures.

Proteomics and metabolomics identify molecular mechanisms of aging potentially predisposing for chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

Proteome profiling in IL-1ß and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis.

Endothelial cells represent major effectors in inflammation and angiogenesis, processes that drive a multitude of pathological states such as atherosclerosis and cancer. Both inflammation and angiogenesis are interconnected with each other in the sense that many pro-inflammatory proteins possess proangiogenic properties and vice versa. To elucidate this interplay further, we present a comparative proteome study of inflammatory and angiogenic activated endothelial cells. HUVEC were stimulated with interleukin 1-ß and VEGF, respectively. Cultured primary cells were fractionated into secreted, cytoplasmic and nuclear protein fractions and processed for subsequent LC-MS/MS analysis. Obtained protein profiles were filtered for fraction-specific proteins to address potential cross fractional contamination, subjected to comparative computational biology analysis (GO-Term enrichment analysis, weighted gene co-expression analysis) and compared to published mRNA profiles of IL-1ß respectively VEGF stimulated HUVEC. GO Term enrichment analysis and comparative pathway analysis revealed features such as NOD and NfkB signaling for inflammatory activated HUVEC and VEGF and ErB signaling for VEGF-activated HUVEC with potential crosstalk via map kinases MAP2K2. Weighted protein co-expression network analysis revealed several potential hub genes so far not associated with driver function in inflammation or angiogenesis such as HSPG2, ANXA3, and GPI. "Classical" inflammation or angiogenesis markers such as IL6, CXCL8 or CST1 were found in a less central position within the co-expression networks. In conclusion, this study reports a framework for the computational biology based analysis of proteomics data applied to cytoplasmic, nucleic and extracellular fractions of quiescent, inflammatory and angiogenic activated HUVEC. Novel potential hub genes relevant for these processes were successfully identified.

Combined Proteome and Eicosanoid Profiling Approach for Revealing Implications of Human Fibroblasts in Chronic Inflammation.

During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.

Evaluation of inflammation-related signaling events covering phosphorylation and nuclear translocation of proteins based on mass spectrometry data.

Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data. SIGNIFICANCE: Relevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma.

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.

Proteomics and transcriptomics of peripheral nerve tissue and cells unravel new aspects of the human Schwann cell repair phenotype.

The remarkable feature of Schwann cells (SCs) to transform into a repair phenotype turned the spotlight on this powerful cell type. SCs provide the regenerative environment for axonal re-growth after peripheral nerve injury (PNI) and play a vital role in differentiation of neuroblastic tumors into a benign subtype of neuroblastoma, a tumor originating from neural crest-derived neuroblasts. Hence, understanding their mode-of-action is of utmost interest for new approaches in regenerative medicine, but also for neuroblastoma therapy. However, literature on human SCs is scarce and it is unknown to which extent human SC cultures reflect the SC repair phenotype developing after PNI in patients. We performed high-resolution proteome profiling and RNA-sequencing on highly enriched human SC and fibroblast cultures, control and ex vivo degenerated nerve explants to identify novel molecules and functional processes active in repair SCs. In fact, we found cultured SCs and degenerated nerves to share a similar repair SC-associated expression signature, including the upregulation of JUN, as well as two prominent functions, i.e., myelin debris clearance and antigen presentation via MHCII. In addition to myelin degradation, cultured SCs were capable of actively taking up cell-extrinsic components in functional phagocytosis and co-cultivation assays. Moreover, in cultured SCs and degenerated nerve tissue MHCII was upregulated at the cellular level along with high expression of chemoattractants and co-inhibitory rather than -stimulatory molecules. These results demonstrate human SC cultures to execute an inherent program of nerve repair and support two novel repair SC functions, debris clearance via phagocytosis-related mechanisms and type II immune-regulation. GLIA 2016;64:2133-2153.

Contribution of Human Fibroblasts and Endothelial Cells to the Hallmarks of Inflammation as Determined by Proteome Profiling.

In order to systematically analyze proteins fulfilling effector functionalities during inflammation, here we present a comprehensive proteome study of inflammatory activated primary human endothelial cells and fibroblasts. Cells were stimulated with interleukin 1-ß and fractionated in order to obtain secreted, cytoplasmic and nuclear protein fractions. Proteins were submitted to a data-dependent bottom up analytical platform using a QExactive orbitrap and the MaxQuant software for protein identification and label-free quantification. Results were further combined with similarly generated data previously obtained from the analysis of inflammatory activated peripheral blood mononuclear cells. Applying a false discovery rate of less than 0.01 at both, peptide and protein level, a total of 8370 protein groups assembled from 117,599 peptides was identified; mass spectrometry data have been made fully accessible via ProteomeXchange with identifier PXD003406 to PXD003417.Comparative proteome analysis allowed us to determine common and cell type-specific inflammation signatures comprising novel candidate marker molecules and related expression patterns of transcription factors. Cardinal features of inflammation such as interleukin 1-ß processing and the interferon response differed substantially between the investigated cells. Furthermore, cells also exerted similar inflammation-related tasks; however, by making use of different sets of proteins. Hallmarks of inflammation thus emerged, including angiogenesis, extracellular matrix reorganization, adaptive and innate immune responses, oxidative stress response, cell proliferation and differentiation, cell adhesion and migration in addition to monosaccharide metabolic processes, representing both, common and cell type-specific responsibilities of cells during inflammation.

Targeting breast cancer-associated fibroblasts to improve anti-cancer therapy.

In recent years, mass spectrometry-based proteomics has undergone significant development steps which may be divided into an exploratory phase, a consolidation phase and an application phase. We are in a stage now where we are able to apply mass spectrometric technologies to answer complex and clinically relevant questions. This is demonstrated here with respect to a current hot topic, namely the consideration of the cancer-supporting microenvironment as a target of new and more efficient anti-cancer therapy. Actually, the relevance of micro environmental stromal cells to tumor initiation and promotion has been clearly recognized. However, the individual kind and degree of stroma-derived tumor promotion can so far hardly be determined in patients, and hardly any therapeutic option exists to dismantle the cancer cells of the stroma-derived support. Quite remarkably, the response of stromal cells to standard chemotherapeutics is also rather unknown. In this Perspective, experimental strategies how to address such issues are outlined in detail. Different cell systems are presented as powerful models which allow identifying relevant marker molecules. Targeted proteomics is presented as method of choice for both, drug screening in vitro as well as monitoring drug responses in patients. By this means, a way of classifying different functional tumor promoting mechanisms, evaluating how current treatment strategies may affect cancer-associated fibroblasts, identifying effective drugs targeting these cancer-associated cells and, may be most importantly, demonstrating how combined therapeutic strategies may improve the efficiency of anti-cancer treatments are indicated.

Quantification of cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters.

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low abundant molecular species still rely on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study, we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama ( www.panoramaweb.org ). Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses, we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005 %, the accuracy was between 80 and 120 %, and the median coefficient of variation for LC/MS was only 2.2 %. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20 % for most peptides. With appropriate marker molecules, we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states, and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls. Graphical Abstract Work flow diagram: Data processing steps beginning with orbitrap-based shotgun data acquisition and MaxQuant data analysis, followed by peptide and transition selection for MRM analysis using Skyline and experimental validation using triple quadrupole MS.

Comprehensive assessment of proteins regulated by dexamethasone reveals novel effects in primary human peripheral blood mononuclear cells.

Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415-23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1ß, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects.