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

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

Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells

Abstract Background In the bone marrow, MSCs reside in a hypoxic milieu (1–5% O2) that is thought to preserve their multipotent state. Typically, in vitro expansion of MSCs is performed under normoxia (~ 21% O2), a process that has been shown to impair their function. Here, we evaluated the characteristics and function of MSCs cultured under hypoxia and hypothesized that, when compared to normoxia, dedicated hypoxia will augment the functional characteristics of MSCs. Methods Human and porcine bone marrow MSCs were obtained from fresh mononuclear cells. The first study evaluated MSC function following both long-term (10 days) and short-term (48 h) hypoxia (1% O2) culture. In our second study, we evaluated the functional characteristics of MSC cultured under short-term 2% and 5% hypoxia. MSCs were evaluated for their metabolic activity, proliferation, viability, clonogenicity, gene expression, and secretory capacity. Results In long-term culture, common MSC surface marker expression (CD44 and CD105) dropped under hypoxia. Additionally, in long-term culture, MSCs proliferated significantly slower and provided lower yields under hypoxia. Conversely, in short-term culture, MSCs proliferated significantly faster under hypoxia. In both long-term and short-term cultures, MSC metabolic activity was significantly higher under hypoxia. Furthermore, MSCs cultured under hypoxia had upregulated expression of VEGF with concomitant downregulation of HMGB1 and the apoptotic genes BCL-2 and CASP3. Finally, in both hypoxia cultures, the pro-inflammatory cytokine, IL-8, was suppressed, while levels of the anti-inflammatories, IL-1ra and GM-CSF, were elevated in short-term hypoxia only. Conclusions In this study, we demonstrate that hypoxia augments the therapeutic characteristics of both porcine and human MSCs. Yet, short-term 2% hypoxia offers the greatest benefit overall, exemplified by the increase in proliferation, self-renewing capacity, and modulation of key genes and the inflammatory milieu as compared to normoxia. These data are important for generating robust MSCs with augmented function for clinical applications