Multiomics surface receptor profiling of the NCI-60 tumor cell panel uncovers novel theranostics for cancer immunotherapy.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICI) has revolutionized cancer therapy. However, therapeutic targeting of inhibitory T cell receptors such as PD-1 not only initiates a broad immune response against tumors, but also causes severe adverse effects. An ideal future stratified immunotherapy would interfere with cancer-specific cell surface receptors only. METHODS: To identify such candidates, we profiled the surface receptors of the NCI-60 tumor cell panel via flow cytometry. The resulting surface receptor expression data were integrated into proteomic and transcriptomic NCI-60 datasets applying a sophisticated multiomics multiple co-inertia analysis (MCIA). This allowed us to identify surface profiles for skin, brain, colon, kidney, and bone marrow derived cell lines and cancer entity-specific cell surface receptor biomarkers for colon and renal cancer. RESULTS: For colon cancer, identified biomarkers are CD15, CD104, CD324, CD326, CD49f, and for renal cancer, CD24, CD26, CD106 (VCAM1), EGFR, SSEA-3 (B3GALT5), SSEA-4 (TMCC1), TIM1 (HAVCR1), and TRA-1-60R (PODXL). Further data mining revealed that CD106 (VCAM1) in particular is a promising novel immunotherapeutic target for the treatment of renal cancer. CONCLUSION: Altogether, our innovative multiomics analysis of the NCI-60 panel represents a highly valuable resource for uncovering surface receptors that could be further exploited for diagnostic and therapeutic purposes in the context of cancer immunotherapy.

Comprehensive Analysis of Human Cytomegalovirus- and HIV-Mediated Plasma Membrane Remodeling in Macrophages.

The plasma membrane (PM) must be overcome by viruses during entry and release. Furthermore, the PM represents the cellular communication compartment and the immune system interface. Hence, viruses have evolved sophisticated strategies to remodel the PM, for instance to avoid immune sensing and clearance of infected cells. We performed a comprehensive analysis of cell surface dysregulation by two human-pathogenic viruses, human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1), in primary macrophages, which are classical antigen-presenting cells and orchestrators of the immune system. Scanning ion conductance microscopy revealed a loss of roughness and an overall smooth phenotype of HCMV-infected macrophages, in contrast to HIV-1 infection. This phenotype was also evident on the molecular level. When we screened for cell surface receptors modulated by HCMV, 42 of 332 receptors tested were up- or downregulated, whereas HIV-1 affected only 7 receptors. In particular CD164, CD84, and CD180 were targeted by HCMV. Mechanistically, HCMV induced transcriptional silencing of these receptors in an interferon (IFN)-independent manner, and expression was reduced not only by lab-adapted HCMV but also by clinical HCMV isolates. Altogether, our plasma membrane profiling of human macrophages provides clues to understand how viruses evade the immune system and identified novel cell surface receptors targeted by HCMV. IMPORTANCE The PM is a key component that viruses have to cope with. It is a barrier for infection and egress and is critically involved in antiviral immune signaling. We hence asked the question how two immunomodulatory viruses, HIV-1 and HCMV, dysregulate this compartment in infected macrophages, relevant in vivo targets of both viruses. We employed a contact-free microscopic technique to image the PM of infected cells and performed a phenotypic flow cytometry-based screen to identify receptor modulations on a molecular level. Our results show that HIV-1 and HCMV differentially manipulate the PM of macrophages. While HIV-1-mediated changes are relatively subtle, HCMV induces major alterations of the PM. We identify novel immune receptors manipulated by HCMV and define mechanisms of how HCMV interferes with receptor expression. Altogether, our study reveals differential strategies of how two human-pathogenic viruses manipulate infected cells and identifies potential novel pathways of HCMV immune evasion.

The Pig PeptideAtlas: A resource for systems biology in animal production and biomedicine.

Biological research of Sus scrofa, the domestic pig, is of immediate relevance for food production sciences, and for developing pig as a model organism for human biomedical research. Publicly available data repositories play a fundamental role for all biological sciences, and protein data repositories are in particular essential for the successful development of new proteomic methods. Cumulative proteome data repositories, including the PeptideAtlas, provide the means for targeted proteomics, system-wide observations, and cross-species observational studies, but pigs have so far been underrepresented in existing repositories. We here present a significantly improved build of the Pig PeptideAtlas, which includes pig proteome data from 25 tissues and three body fluid types mapped to 7139 canonical proteins. The content of the Pig PeptideAtlas reflects actively ongoing research within the veterinary proteomics domain, and this article demonstrates how the expression of isoform-unique peptides can be observed across distinct tissues and body fluids. The Pig PeptideAtlas is a unique resource for use in animal proteome research, particularly biomarker discovery and for preliminary design of SRM assays, which are equally important for progress in research that supports farm animal production and veterinary health, as for developing pig models with relevance to human health research.

Quantitative proteomics and bioinformatic analysis provide new insight into the dynamic response of porcine intestine to Salmonella Typhimurium.

The enteropathogen Salmonella Typhimurium (S. Typhimurium) is the most commonly non-typhoideal serotype isolated in pig worldwide. Currently, one of the main sources of human infection is by consumption of pork meat. Therefore, prevention and control of salmonellosis in pigs is crucial for minimizing risks to public health. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to explore differences in the response to Salmonella in two segment of the porcine gut (ileum and colon) along a time course of 1, 2, and 6 days post infection (dpi) with S. Typhimurium. A total of 298 proteins were identified in the infected ileum samples of which, 112 displayed significant expression differences due to Salmonella infection. In colon, 184 proteins were detected in the infected samples of which 46 resulted differentially expressed with respect to the controls. The higher number of changes in protein expression was quantified in ileum at 2 dpi. Further biological interpretation of proteomics data using bioinformatics tools demonstrated that the expression changes in colon were found in proteins involved in cell death and survival, tissue morphology or molecular transport at the early stages and tissue regeneration at 6 dpi. In ileum, however, changes in protein expression were mainly related to immunological and infection diseases, inflammatory response or connective tissue disorders at 1 and 2 dpi. iTRAQ has proved to be a proteomic robust approach allowing us to identify ileum as the earliest response focus upon S. Typhimurium in the porcine gut. In addition, new functions involved in the response to bacteria such as eIF2 signaling, free radical scavengers or antimicrobial peptides (AMP) expression have been identified. Finally, the impairment at of the enterohepatic circulation of bile acids and lipid metabolism by means the under regulation of FABP6 protein and FXR/RXR and LXR/RXR signaling pathway in ileum has been established for the first time in pigs. Taken together, our results provide a better understanding of the porcine response to Salmonella infection and the molecular mechanisms underlying Salmonella-host interactions.

Large pore dermal microdialysis and liquid chromatography-tandem mass spectroscopy shotgun proteomic analysis: a feasibility study.

BACKGROUND/AIMS: The purpose of the present pilot study was to investigate the feasibility of combining large pore dermal microdialysis with shotgun proteomic analysis in human skin. METHODS: Dialysate was recovered from human skin by 2000 kDa microdialysis membranes from one subject at three different phases of the study; trauma due to implantation of the dialysis device, a post implantation steady-state period, and after induction of vasodilatation and plasma extravasation. For shotgun proteomics, the proteins were extracted and digested with trypsin. Peptides were separated by capillary and nanoflow HPLC systems, followed by tandem mass spectrometry (MS/MS) on a Quadrupole-TOF hybrid instrument. The MS/MS spectra were merged and mapped to a human target protein database to achieve peptide identification and protein inference. RESULTS: Results showed variation in protein amounts and profiles for each of the different sampling phases. The total protein concentration was 1.7, 0.6, and 1.3 mg/mL during the three phases, respectively. A total of 158 different proteins were identified. Immunoglobulins and the major classes of plasma proteins, including proteases, coagulation factors, apolipoproteins, albumins, and complement factors, make up the major load of proteins in all three test conditions. CONCLUSION: Shotgun proteomics allowed the identification of more than 150 proteins in microdialysis samples from human skin. This highlights the opportunities of LC-MS/MS to study the complex molecular interactions in the skin.

Proteomics: a new tool in bovine claw disease research.

Claw horn disruption (CHD) is a common underlying cause of lameness in dairy cattle which leads to compromised animal welfare and production losses. Despite an intense research effort over the last two decades, progress in reducing the prevalence of lameness due to CHD has been limited. In addition to current research strategies there is a need to develop novel approaches and methods that expand understanding of the disease mechanisms involved in CHD. The objectives of the present study were to explore the potential of liquid chromatography tandem mass spectrometry (LC-MS/MS) in mapping protein expression in three different bovine claw tissues, and to provide a relevant functional annotation of the proteins characterized in these tissues. LC-MS/MS was used to characterize protein expression in coronary band skin (C), claw dermal (D) and lamellar (L) tissues from two heifers. A total of 388 different proteins were identified, with 146 proteins available for identification in C, 279 proteins in D and 269 proteins in L. A functional annotation of the identified proteins was obtained using the on-line Blast2GO tool. Three hundred and sixteen of the identified proteins could be subsequently grouped manually to one or more of five major functional groups related to metabolism, cell structure, immunity, apoptosis and angiogenesis. These were chosen to represent basic cell functions and biological processes potentially involved in the pathogenesis of CHD. The LC-MS/MS-based proteomic analysis presented here is the largest published survey, so far, of the bovine claw tissue proteome.

A Bovine PeptideAtlas of milk and mammary gland proteomes.

Proteome information resources of farm animals are lagging behind those of the classical model organisms despite their important biological and economic relevance. Here, we present a Bovine PeptideAtlas, representing a first collection of Bos taurus proteome data sets within the PeptideAtlas framework. This database was built primarily as a source of information for designing selected reaction monitoring assays for studying milk production and mammary gland health, but it has an intrinsic general value for the farm animal research community. The Bovine PeptideAtlas comprises 1921 proteins at 1.2% false discovery rate (FDR) and 8559 distinct peptides at 0.29% FDR identified in 107 samples from six tissues. The PeptideAtlas web interface has a rich set of visualization and data exploration tools, enabling users to interactively mine information about individual proteins and peptides, their prototypic features, genome mappings, and supporting spectral evidence.

Quantotypic properties of QconCAT peptides targeting bovine host response to Streptococcus uberis.

Mammalian host response to pathogens is associated with fluctuations in high abundant proteins in body fluids as well as in regulation of proteins expressed in relatively low copy numbers like cytokines secreted from immune cells and endothelium. Hence, efficient monitoring of proteins associated with host response to pathogens remains a challenging task. In this paper, we present a targeted proteome analysis of a panel of 20 proteins that are widely believed to be key players and indicators of bovine host response to mastitis pathogens. Stable isotope-labeled variants of two concordant proteotypic peptides from each of these 20 proteins were obtained through the QconCAT method. We present the quantotypic properties of these 40 proteotypic peptides and discuss their application to research in host-pathogen interactions. Our results clearly demonstrate a robust monitoring of 17 targeted host-response proteins. Twelve of these were readily quantified in a simple extraction of mammary gland tissues, while the expression levels of the remaining proteins were too low for direct and stable quantification; hence, their accurate quantification requires further fractionation of mammary gland tissues.

Mahalanobis distance screening of Arabidopsis mutants with chlorophyll fluorescence.

Rapid nondestructive screening of mutants is a common step in many research projects in plant biology. Here we report the development of a method that uses kinetic imaging of chlorophyll fluorescence to detect phenotypes that differ from wild-type plants. The method uses multiple fluorescence features simultaneously in order to catch different types of photosynthesis-related mutants with a single assay. The Mahalanobis distance was used to evaluate the degree of similarity in fluorescence features between the wild-type and test plants, and plants differing strongly from the wild-type were classified as mutants. The method was tested on a collection of photosynthesis-related mutants of Arabidopsis thaliana. The plants were evaluated from images in which the color of each pixel depended on the Mahalanobis distance of the fluorescence features. Two parameters of the color-coding procedure were used to adjust the trade-off between detection of true mutants and erratic classification of wild-type plants as mutants. We found that a large percentage of photosynthesis-related mutants can be detected with this method. Scripts for the free statistics software R are provided to facilitate the practical application of the method.

Quantitative milk proteomics--host responses to lipopolysaccharide-mediated inflammation of bovine mammary gland.

Intramammary infusion of lipopolysaccharide (LPS) in cows induces udder inflammation that partly simulates mastitis caused by infection with Gram-negative bacteria. We have used this animal model to characterize the quantitiative response in the milk proteome during the time course before and immediately after the LPS challenge. Milk samples from three healthy cows collected 3 h before the LPS challenge were compared with milk samples collected 4 and 7 h after the LPS challenge, making it possible to describe the inflammatory response of individual cows. Quantitative protein profiles were obtained for 80 milk proteins, of which 49 profiles changed significantly for the three cows during LPS challenge. New information obtained in this study includes the quantified increase of apolipoproteins and other anti-inflammatory proteins in milk, which are important for the cow's ability to balance the immune response, and the upregulation of both complement C3 and C4 indicates that more than one complement pathway could be activated during LPS-induced mastitis. In the future, this analytical approach may provide valuable information about the differences in the ability of individual cows to resist and recover from mastitis.

Tools for computational processing of LC-MS datasets: a user's perspective.

Liquid chromatography-mass spectrometry (LC-MS) profiling of clinical samples for quantifying absolute ion abundances of peptides and proteins has emerged as a promising approach. Quantitation of changes in protein abundance of large number of samples is challenging and requires automatic processing means. The development of data analysis software is laborious and time-consuming. Fortunately, freely available tools have been recently introduced, which incorporate algorithms for visualization and data processing and allow the user to embed external routines for data analysis. A relevant issue related to the design and evaluation of such tools concerns usability. Properties such as easy access, large datasets management, modularity, integration with other tools, etc, are important for performing large-scale integrative data analysis with methods and visual techniques from different (possibly integrated) tools. In this paper, we consider four freely available tools recently introduced in top international journals in order to identify a list of such usability descriptors. We propose 10 descriptors that can be used both as guidelines when developing new tools and as parameters for assessing usability of existing tools. The considered tools show satisfactory usability properties, and the most recent tools exhibit improved flexibility, indicating a trend towards the design of tools that give the user a more central role in the selection, use and integration of methods and tools.