Abstract Increasing evidence highlights the diverse functions of extracellular vesicles (EVs) as intercellular messengers in all facets of cancer progression, pointing towards a potential new avenue in cancer treatment. Limited understanding of tumor-specific deregulations and lack of suitable drug compounds have hampered clinical translation of targeted interference with EV signaling. The most aggressive types of intracranial ependymoma (EPN) in children and adults are resistant to chemotherapy. Despite enormous diagnostic advancements in classification and stratification, EPN lack recurrent effective molecular targets precluding access of affected patients to precision-based oncology therapeutics. Here, we characterized the proteome of ninety-five EPN and five matching healthy control brain tissue samples. Employing a mulit-omic data integration approach, we identified EVs as an excessively augmented pathway on the functional level. Isolation and molecular characterization of EPN EVs combined with single cell sequencing analysis, and ultra-high content imaging unraveled perivascular mesenchymal cell populations as predominant EV originators. Targeting EV pathways through chemical inhibition of sphingomyelinases (SMPD1, SMPD2, SMPD3) including re-purposed FDA approved compounds revealed growth inhibition, reduced invasion capacity in vitro and significant survival probability in vivo. Transcriptomic analysis and scanning electron microscopy delineated transition of mesenchymal-to neuroepithelial subpopulations in cells and patient-derived xenograft (PDX) models of EPN upon reduction of EV release. This study showcases the inhibition of EV signaling as a promising therapeutic strategy against mesenchymal tumor persister cells, which are the suspected origins of repeated EPN relapses. Our study provides a framework to leverage cell state-specific vulnerabilities and drug repurposing to attack the most aggressive cell populations in cancers with unfavorable prognosis.

Infection with certain cutaneous human papillomaviruses (HPV), in conjunction with chronic ultraviolet (UV) exposure, are the major cofactors of non-melanoma skin cancer (NMSC), the most frequent cancer type worldwide. Cutaneous squamous cell carcinomas (SCCs) as well as tumors in general represent three-dimensional entities determined by both temporal and spatial constraints. Whole tissue proteomics is a straightforward approach to understand tumorigenesis in better detail, but studies focusing on different progression states toward a dedifferentiated SCC phenotype on a spatial level are rare. Here, we applied an innovative proteomic workflow on formalin-fixed, paraffin-embedded (FFPE) epithelial tumors derived from the preclinical animal model Mastomys coucha. This rodent is naturally infected with its genuine cutaneous papillomavirus and closely mimics skin carcinogenesis in the context of cutaneous HPV infections in humans. We deciphered cellular networks by comparing diverse epithelial tissues with respect to their differentiation level and infection status. Our study reveals novel regulatory proteins and pathways associated with virus-induced tumor initiation and progression of SCCs. This approach provides the basis to better comprehend the multistep process of skin carcinogenesis.

Abstract INTRODUCTION: Atypical teratoid/rhabdoid tumors (ATRT) represent frequent brain tumors in infants. In recent years, large-scale landscaping efforts on the epigenome and transcriptome of these tumors have unravelled a high degree of heterogeneity and three major molecular subgroups, termed ATRT-TYR, ATRT-SHH,ATRT-MYC, have been identified. The ATRT-proteome, in turn, still represents a largely unchartered territory. METHODS: We have performed a peptide-based screening approach to characterize the proteome of 40 ATRTs and six ATRT cell-lines. All of these samples had matching methylation data available and 28 also corresponding gene expression data. RESULTS: Unsupervised clustering recapitulated the previously described ATRT groups, revealing also a clear split of the SHH-subgroup in a supratentorial (SHH_1) and an infratentorial subgroup (SHH_2). Overall, we identified 7265 proteins, of which 1320 were differentially expressed between the groups, with an enrichment of spliceosome associated terms in SHH_1 and integrins/cell adhesions molecules in SHH_2. ATRT-MYC displayed an overrepresentation of immune cell markers and the TYR subgroup an enrichment of PI3K- as well as mTOR-signaling. Particularly, genes that have previously been described as signature genes for the ATRT-groups such as FABP7 in ATRT-SHH and OTX2 and MITF in ATRT-TYR were among the highly correlating genes that were both expressed in the proteome and the gene expression datasets. On top of this, our analysis revealed highly differentially expressed drug targets such as the tyrosine-kinase MARCKS (overexpressed in ATRT-TYR) not previously identified in ATRT transcriptome data, which warrant investigation by in vitro drug tests. CONCLUSION: Our data reveal the importance of previously described regulatory hubs in the ATRT subgroups, but additionally highlight novel drug targets that merit further exploration. Currently, drug treatment experiments in ATRT cell lines are ongoing to validate these proteins as drug targets, ultimately aiming to establish new therapeutic strategies in this deadly disease.

Abstract INTRODUCTION: The majority of pediatric ependymoma (EPN) comprise either supratentorial EPN characterized by ZFTA-fusions (ST-EPN-ZFTA) or posterior fossa group A EPN (PF-EPN-A), for both of which only limited therapeutic options are available. Because pediatric EPNs have a relatively low mutational burden, identification and characterization of tumor-associated pathways and molecular processes are of critical importance to reveal potential therapeutic targets. Data from previous transcriptional studies and a cross-species in vivo screen implied aberrant vesicular pathways in ST-EPN-ZFTA, prompting further investigation of their putative role in EPN pathogenesis. METHODS: We investigated EPN group-specific differences in extracellular vesicle (EV) biogenesis pathways in human EPN transcriptome and proteome datasets. In addition, we characterized isolated EPN EVs by mass spectrometry. EPN-specific EV cargo was further investigated by immunofluorescence staining and western blotting. This enhanced understanding of EPN vesicular signaling allowed for a pre-selection of inhibitors targeting specific EV biogenesis pathways. In vitro proliferation and invasion assays as well as in vivo treatment studies were performed on EPN model systems. RESULTS: Integration of multi-omic data from both EPN tissues and EPN-EV-associated proteome led to the identification of ST-EPN-ZFTA-specific EV populations. We could spatially map specific EV markers to the perivascular niche that primarily harbors undifferentiated ST-EPN-ZFTA cell populations. Targeting EV biogenesis pathways by inhibiting factors of the lipid metabolism reduced the abundance of released EVs resulting in altered growth behavior and decreased invasion of tumor cells in vitro. In vivo validation of EV release inhibitors in an orthotopic ST-EPN-ZFTA PDX model significantly reduced tumor growth and increased survival. OUTLOOK: In summary, we have leveraged ST-EPN-ZFTA-specific EV pathways as a potential therapeutic vulnerability. Further mechanistic investigations on EPN EV biogenesis, release, or uptake are expected to improve our understanding of the cross-talk between tumor cells and cells of the microenvironment and may lead to potential new therapeutic avenues.

Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2 h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2 h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

Label-free proteomics by data-dependent acquisition enables the unbiased quantification of thousands of proteins, however it notoriously suffers from high rates of missing values, thus prohibiting consistent protein quantification across large sample cohorts. To solve this, we here present IceR (Ion current extraction Re-quantification), an efficient and user-friendly quantification workflow that combines high identification rates of data-dependent acquisition with low missing value rates similar to data-independent acquisition. Specifically, IceR uses ion current information for a hybrid peptide identification propagation approach with superior quantification precision, accuracy, reliability and data completeness compared to other quantitative workflows. Applied to plasma and single-cell proteomics data, IceR enhanced the number of reliably quantified proteins, improved discriminability between single-cell populations, and allowed reconstruction of a developmental trajectory. IceR will be useful to improve performance of large scale global as well as low-input proteomics applications, facilitated by its availability as an easy-to-use R-package.

Abstract Ependymal tumors (EPNs) account for ~10% of all pediatric brain tumors. Supratentorial EPN characterized by RELA fusions (ST-EPN-RELA) and posterior fossa EPN group A (PF-EPN-A) form the two most frequent molecular groups, both of which are associated with poor prognosis and for which only limited therapeutic options are available. Since pediatric EPNs have a relatively low mutational burden, identification and characterization of tumor-associated pathways and molecular processes is of critical importance to inform potential therapeutic targets. Previous transcriptional studies implicated aberrant vesicular pathways in ST-EPN-RELA, prompting further investigation into their putative role in EPN pathogenesis. To this aim, we isolated extracellular vesicles (EVs) of ST-EPN-RELA patient derived cell lines and performed protein mass spectrometry. The specific ST-EPN-RELA EV protein content resembles the parental cells as well as primary tumors. Promising candidates to be transferred by ST-EPN-RELA EVs but not control EVs were associated with unfolded protein response and endoplasmic reticulum stress. When uptaken by recipient cells of the tumor microenvironment, brain endothelial cells or microglia, ST-EPN-RELA EVs induced proliferation and had a chemoattractant effect towards the tumor. ST-EPN-RELA EVs stimulated angiogenesis of brain endothelial cells potentially by the transfer of ER stress proteins. Uptake of ST-EPN-RELA EVs by microglia changed their activation status indicating a tumor promoting function through EV transfer. Therefore, we hypothesize that vesicular pathways play an important role in the pathogenesis of pediatric ST-EPN-RELAs and that an improved understanding may promote new therapeutic opportunities.

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non-clinical applications.

Post-translational modifications such as phosphorylation can have profound effects on the physicochemical and biological properties of proteins. However, high-throughput and systematic approaches have not yet been developed to assess the effects of specific modification types and sites on protein lifetime, which represents a key parameter for understanding signaling rewiring and drug development. Here we describe a proteomic method, DeltaSILAC, to quantify the impact of site-specific phosphorylation on the endurance of thousands of proteins in live cells. Being configured on the reproducible data-independent acquisition mass spectrometry (DIA-MS), the pulse labeling approach using stable isotope-labeled amino acids in cells (SILAC), together with a novel peptide-level matching strategy, this multiplexed assay revealed the global delaying effect of phosphorylation on protein turnover in growing cancer cells. Further, we identified local sequence and structural features in proximity to the phosphorylated sites that could be associated with protein endurance alterations. We found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness and evolutionarily conserved. DeltaSILAC provides a generalizable approach for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology, which is highly complementary to existing methods. Finally, DeltaSILAC is widely applicable to diverse post-translational modification types and different cell systems.

Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.

Background Posterior fossa A (PFA) ependymomas are one of 9 molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis, and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of chromosome X open reading frame 67 (CXorf67) as a hallmark of PFA ependymoma and showed that CXorf67 can interact with enhancer of zeste homolog 2 (EZH2), thereby inhibiting polycomb repressive complex 2 (PRC2), but the mechanism of action remained unclear. Methods We performed mass spectrometry and peptide modeling analyses to identify the functional domain of CXorf67 responsible for binding and inhibition of EZH2. Our findings were validated by immunocytochemistry, western blot, and methyltransferase assays. Results We find that the inhibitory mechanism of CXorf67 is similar to diffuse midline gliomas harboring H3K27M mutations. A small, highly conserved peptide sequence located in the C-terminal region of CXorf67 mimics the sequence of K27M mutated histones and binds to the SET domain (Su(var)3-9/enhancer-of-zeste/trithorax) of EZH2. This interaction blocks EZH2 methyltransferase activity and inhibits PRC2 function, causing de-repression of PRC2 target genes, including genes involved in neurodevelopment. Conclusions Expression of CXorf67 is an oncogenic mechanism that drives H3K27 hypomethylation in PFA tumors by mimicking K27M mutated histones. Disrupting the interaction between CXorf67 and EZH2 may serve as a novel targeted therapy for PFA tumors but also for other tumors that overexpress CXorf67. Based on its function, we have renamed CXorf67 as “EZH Inhibitory Protein” (EZHIP).

Posterior fossa A (PFA) ependymomas comprise one out of nine molecular groups of ependymoma. PFA tumors are mainly diagnosed in infants and young children, show a poor prognosis and are characterized by a lack of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark. Recently, we reported overexpression of CXorf67 as hallmark of PFA ependymoma and that CXorf67 can interact with polycomb repressive complex 2 (PRC2) thereby inhibiting EZH2. However, a detailed description of the functional domains of CXorf67 and the exact mechanism of EZH2 inhibition is still lacking. We now have performed mass spectrometry (MS) analyses of different CXorf67 protein truncates to identify functional domains in the CXorf67 protein involved in the binding of PRC2 core components. Our results show that PRC2 components like EZH2 and SUZ12 specifically bind to the C-terminal part of the CXorf67 protein. Overexpression of this C-terminal part of CXorf67 was sufficient to induce a strong downregulation of H3K27me3 in HEK293 cell lines. Upon inspection of the amino acid sequence in the C-terminus we discovered that a small, highly conserved amino acid sequence is responsible for binding and inhibition of EZH2 resulting in the perturbation of PRC2 function. A synthetic 21 amino acids long peptide containing these conserved amino acids completely abolished PRC2 activity in a methyltransferase assay. Strikingly, in silico analyses showed that the CXorf67 peptide mimics the H3K27M peptide and both can bind to the SET domain of EZH2, suggesting that the mechanism of EZH2 inhibition by CXorf67 was highly similar to the mechanism observed in pediatric high-grade gliomas harboring H3K27M mutations. Taken together, our findings shed light on the functional domains of CXorf67 and reveal a crucial oncogenic mechanism that may be exploited for targeted therapy in PFA ependymoma.

Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.

The independent reproduction of research results is a cornerstone of experimental research, yet it is beset by numerous challenges, including the quality and veracity of reagents and materials. Much of life science research depends on life materials, including human tissue culture cells. In this study we aimed at determining the degree of variability in the molecular makeup and the ensuing phenotypic consequences in commonly used human tissue culture cells. We collected 14 stock HeLa aliquots from 13 different laboratories across the globe, cultured them in uniform conditions and profiled the genome-wide copy numbers, mRNAs, proteins and protein turnover rates via genomic techniques and SWATH mass spectrometry, respectively. We also phenotyped each cell line with respect to the ability of transfected Let7 mimics to modulate Salmonella infection. We discovered significant heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto variety. We also observed progressive divergence within a specific cell line over 50 successive passages. From the aggregate multi-omic datasets we quantified the response of the cells to genomic variability across the transcriptome and proteome. We discovered organelle-specific proteome remodeling and buffering of protein abundance by protein complex stoichiometry, mediated by the adaptation of protein turnover rates. By associating quantitative proteotype and phenotype measurements we identified protein patterns that explained the varying response of the different cell lines to Salmonella infection. Altogether the results indicate a striking degree of genomic variability, the rapid evolution of genomic variability in culture and its complex translation into distinctive expressed molecular and phenotypic patterns. The results have broad implications for the interpretation and reproducibility of research results obtained from HeLa cells and provide important basis for a general discussion of the value and requirements for communicating research results obtained from human tissue culture cells.

Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.