Assessment of a Large-Scale Unbiased Malignant Pleural Effusion Proteomics Study of a Real-Life Cohort

Background: Pleural effusion (PE) is common in advanced-stage lung cancer patients and is related to poor prognosis. Identification of cancer cells is the standard method for the diagnosis of a malignant PE (MPE). However, it only has moderate sensitivity. Thus, more sensitive diagnostic tools are urgently needed. Methods: The present study aimed to discover potential protein targets to distinguish malignant pleural effusion (MPE) from other non-malignant pathologies. We have collected PE from 97 patients to explore PE proteomes by applying state-of-the-art liquid chromatography-mass spectrometry (LC-MS) to identify potential biomarkers that correlate with immunohistochemistry assessment of tumor biopsy or with survival data. Functional analyses were performed to elucidate functional differences in PE proteins in malignant and benign samples. Results were integrated into a clinical risk prediction model to identify likely malignant cases. Sensitivity, specificity, and negative predictive value were calculated. Results: In total, 1689 individual proteins were identified by MS-based proteomics analysis of the 97 PE samples, of which 35 were diagnosed as malignant. A comparison between MPE and benign PE (BPE) identified 58 differential regulated proteins after correction of the p-values for multiple testing. Furthermore, functional analysis revealed an up-regulation of matrix intermediate filaments and cellular movement-related proteins. Additionally, gene ontology analysis identified the involvement of metabolic pathways such as glycolysis/gluconeogenesis, pyruvate metabolism and cysteine and methionine metabolism. Conclusion: This study demonstrated a partial least squares regression model with an area under the curve of 98 and an accuracy of 0.92 when evaluated on the holdout test data set. Furthermore, highly significant survival markers were identified (e.g., PSME1 with a log-rank of 1.68 × 10−6 ).info:eu-repo/semantics/publishedVersio

Meta-Analysis of MS-Based Proteomics Studies Indicates Interferon Regulatory Factor 4 and Nucleobindin1 as Potential Prognostic and Drug Resistance Biomarkers in Diffuse Large B Cell Lymphoma

Funding: Rune Matthiesen is supported by Fundação para a Ciência e a Tecnologia (CEEC position, 2019–2025 investigator). This article is a Fiigureresult of the projects (iNOVA4Health— UIDB/04462/2020), supported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT—Portuguese Foundation for Science and Technology under the project numbers: PTDC/BTM-TEC/30087/2017 and PTDC/BTM-TEC/30088/2017.The prognosis of diffuse large B cell lymphoma (DLBCL) is inaccurately predicted using clinical features and immunohistochemistry (IHC) algorithms. Nomination of a panel of molecules as the target for therapy and predicting prognosis in DLBCL is challenging because of the divergences in the results of molecular studies. Mass spectrometry (MS)-based proteomics in the clinic represents an analytical tool with the potential to improve DLBCL diagnosis and prognosis. Previous proteomics studies using MS-based proteomics identified a wide range of proteins. To achieve a consensus, we reviewed MS-based proteomics studies and extracted the most consistently significantly dysregulated proteins. These proteins were then further explored by analyzing data from other omics fields. Among all significantly regulated proteins, interferon regulatory factor 4 (IRF4) was identified as a potential target by proteomics, genomics, and IHC. Moreover, annexinA5 (ANXA5) and nucleobindin1 (NUCB1) were two of the most up-regulated proteins identified in MS studies. Functional enrichment analysis identified the light zone reactions of the germinal center (LZ-GC) together with cytoskeleton locomotion functions as enriched based on consistent, significantly dysregulated proteins. In this study, we suggest IRF4 and NUCB1 proteins as potential biomarkers that deserve further investigation in the field of DLBCL sub-classification and prognosis.publishersversionpublishe

Assessment of a Large-Scale Unbiased Malignant Pleural Effusion Proteomics Study of a Real-Life Cohort

Funding Information: R.M. is supported by Fundação para a Ciência e a Tecnologia (CEEC position, 2019–2025 investigator). This article is a result of the projects (iNOVA4Health—UIDB/04462/2020), supported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT—Portuguese Foundation for Science and Technology under the projects number PTDC/BTM-TEC/30087/2017 and PTDC/BTM-TEC/30088/2017. Publisher Copyright: © 2022 by the authors.Background: Pleural effusion (PE) is common in advanced-stage lung cancer patients and is related to poor prognosis. Identification of cancer cells is the standard method for the diagnosis of a malignant PE (MPE). However, it only has moderate sensitivity. Thus, more sensitive diagnostic tools are urgently needed. Methods: The present study aimed to discover potential protein targets to distinguish malignant pleural effusion (MPE) from other non-malignant pathologies. We have collected PE from 97 patients to explore PE proteomes by applying state-of-the-art liquid chromatography-mass spectrometry (LC-MS) to identify potential biomarkers that correlate with immunohistochemistry assessment of tumor biopsy or with survival data. Functional analyses were performed to elucidate functional differences in PE proteins in malignant and benign samples. Results were integrated into a clinical risk prediction model to identify likely malignant cases. Sensitivity, specificity, and negative predictive value were calculated. Results: In total, 1689 individual proteins were identified by MS-based proteomics analysis of the 97 PE samples, of which 35 were diagnosed as malignant. A comparison between MPE and benign PE (BPE) identified 58 differential regulated proteins after correction of the p-values for multiple testing. Furthermore, functional analysis revealed an up-regulation of matrix intermediate filaments and cellular movement-related proteins. Additionally, gene ontology analysis identified the involvement of metabolic pathways such as glycolysis/gluconeogenesis, pyruvate metabolism and cysteine and methionine metabolism. Conclusion: This study demonstrated a partial least squares regression model with an area under the curve of 98 and an accuracy of 0.92 when evaluated on the holdout test data set. Furthermore, highly significant survival markers were identified (e.g., PSME1 with a log-rank of 1.68 × 10−6).publishersversionpublishe

Methods of Liver Stem Cell Therapy in Rodents as Models of Human Liver Regeneration in Hepatic Failure

Cell therapy is a promising intervention for treating liver diseases and liver failure. Different animal models of human liver cell therapy have been developed in recent years. Rats and mice are the most commonly used liver failure models. In fact, rodent models of hepatic failure have shown significant improvement in liver function after cell infusion. With the advent of stem-cell technologies, it is now possible to re-programme adult somatic cells such as skin or hair-follicle cells from individual patients to stem-like cells and differentiate them into liver cells. Such regenerative stem cells are highly promising in the personalization of cell therapy. The present review article will summarize current approaches to liver stem cell therapy with rodent models. In addition, we discuss common cell tracking techniques and how tracking data help to direct liver cell therapy research in animal models of hepatic failure

Proteomic landscape of extracellular vesicles for diffuse large b‐cell lymphoma subtyping

Funding Information: R.M. is supported by Funda??o para a Ci?ncia e a Tecnologia (CEEC position, 2019?2025 investigator). This article is a result of the projects (iNOVA4Health?UID/Multi/04462/2013), supported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT?Portuguese Foundation for Science and Technology under the projects number PTDC/BTM?TEC/30087/2017 and PTDC/BTM?TEC/30088/2017. B.C.S. is supported by the Cham-palimaud Foundation and the EMBO Installation Grant 3921. Funding Information: Funding: R.M. is supported by Fundação para a Ciência e a Tecnologia (CEEC position, 2019–2025 investigator). This article is a result of the projects (iNOVA4Health—UID/Multi/04462/2013), sup‐ ported by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work is also funded by FEDER funds through the COMPETE 2020 Programme and National Funds through FCT—Portuguese Foundation for Science and Technology under the projects number PTDC/BTM‐TEC/30087/2017 and PTDC/BTM‐TEC/30088/2017. B.C.S. is supported by the Cham‐ palimaud Foundation and the EMBO Installation Grant 3921. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.The role of extracellular vesicles (EVs) proteome in diffuse large B‐cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state‐of‐the‐art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole‐cell and secreted EVs proteomes of the two cell‐of‐origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole‐cell proteome and 228/608 proteins from EVs (adjust p‐value < 0.05/p‐value < 0.05). In our preclinical model system, we demonstrated that the EV prote-ome and the whole‐cell proteome possess the capacity to separate cell lines into ABC and GCB sub-types. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B‐cell receptor (BCR), Fc_gamma R‐mediated phagocytosis, ErbB signaling, and endocyto-sis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow‐up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.publishersversionpublishe

Evaluating the Proliferation of Human PeripheralBlood Mononuclear Cells Using MTT Assay

Introduction: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a safe, convenient, and low-cost technique with high applications for the measurement of cell proliferation rate in researches and clinical laboratories. Our aim was to evaluate the proliferation rate of human peripheral blood mononuclear cells (PBMCs) and production rate of Tumor necrosis factor alpha (TNF-α) by these cells after various mitogens stimulation in different situations. Methods: The MTT test was performed with various concentrations of mitogens including concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA) on the PBMCs. The cells were incubated for 24, 48, 72, and 96 hours in the culture medium and TNF-α cytokine assay was performed on the supernatant of the cultured splenocytes using the enzyme-linked immunosorbent assay (ELISA) method. Results: The optimal time and incubation of the PBMCs with the mixture of PHA-ConA were 5 μg/mL and 72 hours, respectively. The TNF-α level increased significantly after PHA-ConA and PHA stimulation. Conclusion: The results showed that the mixture of PHA-ConA (at the concentration of 5 μg/mL) can give rise to the optimal results on stimulation of the PBMcs using the MTT assay after 72 hours incubation

The Effects of Hypoxia on U937 Cell Line in Mesenchymal Stem Cells Co-Culture System

Purpose: Mesenchymal Stem Cells (MSCs) are the most important members of Bone Marrow (BM) milieu. MSCs affect different kinds of cells, particularly malignant cells of hematologic malignancies, but the effects of MSCs are unclear exactly. Here we analyzed the effects of derived Umbilical Cord Blood-MSCs on proliferation, cell death and some surface markers of U937 cell line in a Co-culture system with MSCs. Methods: Here we designed Co-culture systems as a model of BM milieu. We cultured U937 cells on UCB-MSCs and MSCs Conditioned Medium (C.M) driven and then treated U937 cells with optimum concentration of chloride cobalt (CoCl2) as a hypoxia-mimetic agent. In addition, we applied suitable concentrations of H2O2 to induce cell death. Proliferation rate, cell death rate and some surface markers of hypoxic U937 cells were analyzed by MTT assay, flow cytometry and Real Time-PCR were flown respectively. Results: UCB-MSCs showed supportive effects on U937 proliferation rate in normoxia and hypoxia. Lethal effect of H2O2 suppressed in the presence of UCB-MSCs in hypoxia and normoxia. Among CD11a, CD14, CD49d, CD54 and CD116 markers, CD49d was down regulated in presence of UCB-MSCs and CD116 was up regulated in hypoxia. Other markers didn’t show any significant changes. Conclusion: This work provides evidences that MSCs play critical roles in U937 cells biology. These observations shed new light on MSCs roles and demonstrated that MSCs should be regarded as an important member of BM milieu in several clinical applications such as BM transplantation prognosis and treatment of hematologic malignancies