Platform independent protein-based cell-of-origin subtyping of diffuse large B-cell lymphoma in formalin-fixed paraffin-embedded tissue

Diffuse large B-cell lymphoma (DLBCL) is commonly classified by gene expression profiling according to its cell of origin (COO) into activated B-cell (ABC)-like and germinal center B-cell (GCB)-like subgroups. Here we report the application of label-free nano-liquid chromatography - Sequential Window Acquisition of all THeoretical fragment-ion spectra - mass spectrometry (nanoLC-SWATH-MS) to the COO classification of DLBCL in formalin-fixed paraffin-embedded (FFPE) tissue. To generate a protein signature capable of predicting Affymetrix-based GCB scores, the summed log(2)-transformed fragment ion intensities of 780 proteins quantified in a training set of 42 DLBCL cases were used as independent variables in a penalized zero-sum elastic net regression model with variable selection. The eight-protein signature obtained showed an excellent correlation (r=0.873) between predicted and true GCB scores and yielded only 9 (21.4%) minor discrepancies between the three classifications: ABC, GCB, and unclassified. The robustness of the model was validated successfully in two independent cohorts of 42 and 31 DLBCL cases, the latter cohort comprising only patients aged >75 years, with Pearson correlation coefficients of 0.846 and 0.815, respectively, between predicted and NanoString nCounter based GCB scores. We further show that the 8-protein signature is directly transferable to both a triple quadrupole and a Q Exactive quadrupole-Orbitrap mass spectrometer, thus obviating the need for proprietary instrumentation and reagents. This method may therefore be used for robust and competitive classification of DLBCLs on the protein level

Tachycardiomyopathy entails a dysfunctional pattern of interrelated mitochondrial functions

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH–iPSC–CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm2 vs. 538.9 ± 23.8 µm2, p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH–iPSC–CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O2·s−1·mg−1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies

Shear Force Processing of Lipoaspirates for Stem Cell Enrichment Does Not Affect Secretome of Human Cells Detected by Mass Spectrometry In Vitro

Background: Lipofilling is one of the most often performed surgical procedures in plastic and reconstructive surgery. Lipoaspirates provide a ready source of stem cells and secreted factors that contribute to neoangiogenesis and fat graft survival. However, the regulations about the enrichment of these beneficial cells and factors are ambiguous. In this study, the authors tested whether a combination of centrifugation and homogenization allowed the enrichment of viable stem cells in lipoaspirates through the selective removal of tumescent solution, blood, and released lipids without significantly affecting the cell secretome. Methods: Human lipoaspirate was harvested from six different patients using water jet-assisted liposuction. Lipoaspirate was homogenized by first centrifugation (3584 rpm for 2 minutes), shear strain (10 times intersyringe processing), and second centrifugation (3584 rpm for 2 minutes). Stem cell enrichment was shown by cell counting after stem cell isolation. Lipoaspirate from different processing steps (unprocessed, after first centrifugation, after homogenization, after second centrifugation) was incubated in serum-free cell culture medium for mass spectrometric analysis of secreted proteins. Results: Lipoaspirate homogenization leads to a significant 2.6 +/- 1.75-fold enrichment attributable to volume reduction without reducing the viability of the stem cells. Protein composition of the secretome did not change significantly after tissue homogenization. Considering the enrichment effects, there were no significant differences in the protein concentration of the 83 proteins found in all processing steps. Conclusions: Stem cells can be enriched mechanically without significantly affecting the composition of secreted proteins. Shear-assisted enrichment of lipoaspirate constitutes no substantial manipulation of the cells' secretome

Deregulation of protein methylation in melanoma

Loss of methylthioadenosine phosphorylase (MTAP) expression and a concomitant accumulation of 5'-methyl-thioadenosine (MTA) characterise several tumour entities including malignant melanoma. MTA affects cellular signalling, proliferation and migration not only of cancer but also surrounding cells including lymphocytes and stromal fibroblasts. The mode of action of MTA is still not known. Interestingly, MTA is a known potent inhibitor of protein arginine methyltransferases (PRMTs) and is used as a tool in studying activity and impact of PRMTs. This study aimed at analysing PRMTs in melanoma and the potential impact of MTA on tumourigenesis. Our findings demonstrate that expression of PRMT4/CARM1 and PRMT6 is deregulated in melanoma, whereas expression of the remaining PRMTs stays unchanged. General PRMT activity and, consequently, symmetric and asymmetric protein methylation are reduced significantly in melanoma cells and tissues. This is due to a loss of MTAP expression and accumulation of MTA. Reduction of protein methylation by MTA affects cell signalling and leads, for example, to an activation of extracellular signal-regulated kinase (ERK) activity. The effects of endogeneous MTA on PRMTs as presented in this study can strongly support the migratory and invasive phenotype of melanoma cells

Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma

<div><p>Deficiency of methylthioadenosine phosphorylase (<i>MTAP</i>) supports melanoma development and progression through accumulation of its substrate 5’-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in <i>MTAP</i> may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for <i>MTAP</i>. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP.</p></div

Enzyme kinetics of both <i>in vitro</i>-translated MTAP variants.

<p>(A) Western Blot for determination of MTAP expression by <i>in vitro</i> transcription/translation of the pCMX-PL1 (1.0) control plasmid and the expression constructs for MTAP-56I (2.56-fold increased) and MTAP-56V (2.28-fold increased). Densitometric quantification revealed increased MTAP amount. The original Western blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s001" target="_blank">S1 Fig</a>. (B) Analysis of the metabolic activity of the <i>in vitro</i>-translated products by liquid chromatography–tandem mass spectrometry. Catalytic rates of IVT-MTAP-56I and IVT-MTAP-56V were corrected for the rate of the control, which was set at 1.</p

Effect of both MTAP variants in transiently cells.

<p>Quantification of MTAP expression at the mRNA (A) and protein level (B) in the melanoma cell line Mel Juso after transient transfection of expression constructs for MTAP-56I and MTAP-56V or control plasmid (pCMX-PL1). The original Western blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s002" target="_blank">S2 Fig</a>. (C) Analysis of intracellular MTA levels in the transiently transfected Mel Juso cells.</p

Confirmation of cDNA genotype by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based protein expression analysis.

<p>Sole expression of the 56I-allele was observed in melanoma cell lines Mel Im, Mel Ho, and A375, whereas the 56V-allele was detected exclusively in WM793, WM1366, and WM293A. In the fibroblast cell line 3F0379, both alleles were co-expressed equally.</p

Enzyme kinetics of both MTAP variants in stably transfected cells.

<p>Quantification of MTAP expression at the mRNA (A) and protein level (B) in the melanoma cell line Mel Juso after stable transfection of control plasmid (pCMX-PL1) or expression constructs for MTAP-56I and MTAP-56V. The original Western blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s003" target="_blank">S3 Fig</a>. (C) Analysis of intracellular MTA levels in the stably transfected Mel Juso cell clones.</p