Secretome Proteomics Research Articles

SWATH-MS Based Secretome Proteomic Analysis of Pseudomonas aeruginosa Against MRSA.

The study uses Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH)-MS in conjunction with secretome proteomics to identify key proteins that Pseudomonas aeruginosa secretes against methicillin-resistant Staphylococcus aureus (MRSA). Variations in the inhibition zones indicated differences in strain resistance. Multivariate statistical methods were applied to filter the proteomic results, revealing five potential protein biomarkers, including Peptidase M23. Gene ontology (GO) analysis and sequence alignment supported their antibacterial activity. Thus, SWATH-MS provides a comprehensive understanding of the secretome of P. aeruginosa in its action against MRSA, guiding future antibacterial research.

Comparative proteomic profiling of the ovine and human PBMC inflammatory response

Understanding the cellular and molecular mechanisms of inflammation requires robust animal models. Sheep are commonly used in immune-related studies, yet the validity of sheep as animal models for immune and inflammatory diseases remains to be established. This cross-species comparative study analyzed the in vitro inflammatory response of ovine (oPBMCs) and human PBMCs (hPBMCs) using mass spectrometry, profiling the proteome of the secretome and whole cell lysate. Of the entire cell lysate proteome (oPBMCs: 4217, hPBMCs: 4574 proteins) 47.8% and in the secretome proteome (oPBMCs: 1913, hPBMCs: 1375 proteins) 32.8% were orthologous between species, among them 32 orthologous CD antigens, indicating the presence of six immune cell subsets. Following inflammatory stimulation, 71 proteins in oPBMCs and 176 in hPBMCs showed differential abundance, with only 7 overlapping. Network and Gene Ontology analyses identified 16 shared inflammatory-related terms and 17 canonical pathways with similar activation/inhibition patterns in both species, demonstrating significant conservation in specific immune and inflammatory responses. However, ovine PMBCs also contained a unique WC1+γδ T-cell subset, not detected in hPBMCs. Furthermore, differences in the activation/inhibition trends of seven canonical pathways and the sets of DAPs between sheep and humans, emphasize the need to consider interspecies differences in translational studies and inflammation research.

Secretome processing for proteomics: A methods comparison.

The cancer cell secretome comprises a treasure-trove for biomarkers since it reflects cross-talk between tumor cells and their surrounding environment with high detectability in biofluids. In this study, we evaluated six secretome sample processing workflows coupled to single-shot mass spectrometry: (1) Protein concentration by ultrafiltration with a molecular weight cut-off (MWCO) filter and sample preparation through in-gel digestion (IGD); (2) Acetone protein precipitation coupled to IGD; (3) MWCO filter-based protein concentration followed by to in-solution digestion (ISD); (4) Acetone protein precipitation coupled to ISD; (5) Direct ISD; (6) Secretome lyophilization and ISD. To this end, we assessed workflow triplicates in terms of total number of protein identifications, unique identifications, reproducibility of protein identification and quantification and detectability of small proteins with important functions in cancer biology such as cytokines, chemokines, and growth factors. Our findings revealed that acetone protein precipitation coupled to ISD outperformed the other methods in terms of the number of identified proteins (2246) and method reproducibility (correlation coefficient between replicates (r=0.94, CV=19%). Overall, especially small proteins such as those from the classes mentioned above were better identified using ISD workflows. Concluding, herein we report that secretome protein precipitation coupled to ISD is the method of choice for high-throughput secretome proteomics via single shot nanoLC-MS/MS.

Applying single-cell highly multiplexed secretome proteomics to characterize immunotherapeutic products and predict clinical responses.

Genetically and phenotypically identical immune cell populations can be highly heterogenous in terms of their immune functions and protein secretion profiles. The microfluidic chip-based single-cell highly multiplexed secretome proteomics enables characterization of cellular heterogeneity of immune responses at different cellular and molecular layers. Increasing evidence has demonstrated that polyfunctional T cells that simultaneously produce 2+ proteins per cell at the single-cell level are key effector cells that contribute to the development of potent and durable cellular immunity against pathogens and cancers. The functional proteomic technology offers a wide spectrum of cellular function assessment and can uniquely define highly polyfunctional cell subsets with cytokine signatures from live individual cells. This high-dimensional single-cell analysis provides deep dissection into functional heterogeneity and helps identify predictive biomarkers and potential correlates that are crucial for immunotherapeutic product design optimization and personalized immunotherapy development to achieve better clinical outcomes.

Mycelial and secretome proteomic dynamics of L. squarrosulus AF5 in azo dye degradation

Mycoremediation of textile effluents is a practical and eco-friendly approach for mineralizing synthetic dyes. The textile, food processing, chemical, leather, dyestuff, dyeing, and pharmaceutical industries discharge significant amounts of effluents including different types of dyes. One of the largest manufacturers of dyes is the textile industry, and dyes discharged from these industries directly affect the natural ecosystem by entering in to the soil and water. These industries release effluents that are carcinogenic and pose a potential threat to flora and fauna. Distinct fungal species are identified as dye decomposers because these can produce a variety of extracellular enzymes involved in catabolism. The strain isolated from the dye effluents was identified as Lentinus squarrosulus AF5 and had a remarkable ability for decolorization (50–96 %) within 72 h using a dye mix consisting of AB10B, RB5, and RB160 (500 mg L-1. The dynamics of the protein profile was deciphered using gel-free/label-free nLC-MS/MS analysis. Distinct protein profiles in the mycelial extract of Lentinus squarrosulus AF5 were observed following exposure of the strain with dyes mix. About 90 proteins have been depicted by proteomic analysis wherein 10 proteins appear to be differentially expressed during dye treated conditions. Secretome proteome had denoted 13 proteins under similar conditions. Notable levels of manganese peroxidase (258.84 ± 0.001 U/ml) along with relatively lower levels of lignin peroxidases (194.98 ± 0.002 U/ml) and laccase (134.33 ± 0.007 U/ml) were detected during dye treated condition, suggesting thereby the extracellular peroxidases displaying a pivotal role in decolorization process. The UV-Visible, FTIR spectroscopic along with HPLC and LC-QTOF-MS analyses of the dye supernatants following incubation with Lentinus squarrosulus AF5 had indicated the degradation of the dyes. These observations suggest Lentinus squarrosulus AF5 to be a potential strain for the catabolism of azo dyes.

Abstract 015: Hypertensive Stretch On The Regulation Of The Immunological Synapse And Secretomes Of Male And Female Renal Endothelial Cells

Immune cells and inflammation contribute to the development of hypertension and renal injury. While endothelial cells are recognized as professional antigen presenting cells, they can express both Major Histocompatibility Complex (MHC) I and II and possess antigen-presenting functions. Endothelial cell activation and inflammation are critical in the development of hypertension. However, the mechanisms by which inflammatory hypertensive stimuli regulate renal endothelial immunological synapse proteins remains unknown. We hypothesize that inflammatory hypertensive mechanical stretch promotes the regulation of MHC I and II and costimulatory molecules on mouse renal endothelial cells (MRECs) in a sex-dependent manner. MRECs derived from both sexes underwent normotensive (5%; 1 Hz), hypertensive (10%, 1 Hz), and supra-hypertensive (15% 1 Hz) uniaxial cyclical stretch for 48 hours. We then assessed CD80, CD86, MHC II, MHC I, CD70, and CD40 by immunoblot analysis. A proteome multiplex assay was used to analyze male and female MREC secretomes during 5%, 10%, and 15% stretch. Hypertensive 10% stretch significantly increased only CD40 expression in males, while it upregulated CD80 and CD70 expression in females (p<0.05). Under 15% stretch, males significantly upregulated MHC I, CD70, and CD40 compared to I-Ab, CD80, CD86, and CD70 in females MRECs (p<0.05). We investigated sex differences and found that females significantly increased CD70 in both 10% and 15% stretch compared to males MRECs (p<0.05). We compared sex differences from our secretome proteomics in male and female MRECs at 10% and 15% stretch. We found that pathways associated with wound healing, Th17 activation, IL-6, and NF-κB signaling were upregulated in male MRECs undergoing 10% stretch. Interestingly, Th17 activation, natural killer cell signaling, and IL-23 signaling were downregulated in female MRECs undergoing 10% stretch. Under 15% stretch, IL-13 signaling, HMBG1 signaling, B cell signaling, and IL-13 signaling were downregulated in MRECs from both sexes. Our data provides novel insights into how inflammatory hypertensive stretch regulates immunological synapse proteins and secretomes in renal endothelial cells by stretch and sex-dependent manner.

Exploring Visceral and Subcutaneous Adipose Tissue Secretomes in Human Obesity: Implications for Metabolic Disease.

Adipose tissue secretions are depot-specific and vary based on anatomical location. Considerable attention has been focused on visceral (VAT) and subcutaneous (SAT) adipose tissue with regard to metabolic disease, yet our knowledge of the secretome from these depots is incomplete. We conducted a comprehensive analysis of VAT and SAT secretomes in the context of metabolic function. Conditioned media generated using SAT and VAT explants from individuals with obesity were analyzed using proteomics, mass spectrometry, and multiplex assays. Conditioned media were administered in vitro to rat hepatocytes and myotubes to assess the functional impact of adipose tissue signaling on insulin responsiveness. VAT secreted more cytokines (IL-12p70, IL-13, TNF-α, IL-6, and IL-8), adipokines (matrix metalloproteinase-1, PAI-1), and prostanoids (TBX2, PGE2) compared with SAT. Secretome proteomics revealed differences in immune/inflammatory response and extracellular matrix components. In vitro, VAT-conditioned media decreased hepatocyte and myotube insulin sensitivity, hepatocyte glucose handling, and increased basal activation of inflammatory signaling in myotubes compared with SAT. Depot-specific differences in adipose tissue secretome composition alter paracrine and endocrine signaling. The unique secretome of VAT has distinct and negative impact on hepatocyte and muscle insulin action.

Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata.

Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host–parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host–parasite interaction within the horse host.

Ex vivo glucocorticoid-induced secreted proteome approach for discovery of glucocorticoid-responsive proteins in human serum.

To identify glucocorticoid-responsive proteins measurable in human serum that may have clinical utility in therapeutic drug monitoring and the diagnosis of cortisol excess or deficiency. A phased biomarker discovery strategy was conducted in two cohorts. Secretome from peripheral blood mononuclear cells (PBMC) isolated from six volunteers after ex vivo incubation ± dexamethasone (DEX) 100ng/mL for 4 h and 24 h was used for candidate discovery and qualification using untargeted proteomics and a custom multiple reaction monitoring mass spectrometry (MRM-MS) assay, respectively. For validation, five candidates were measured by immunoassay in serum from an independent cohort (n=20), sampled at 1200 h before and after 4mg oral DEX. The discovery secretome proteomics data generated a shortlist of 45 candidates, with 43 measured in the final MRM-MS assay. Differential analysis revealed 16 proteins that were significant in at least one of two time points. In the validation cohort, 3/5 serum proteins were DEX-responsive, two significantly decreased: lysozyme C (p<0.0001) and nucleophosmin-1 (p<0.01), while high mobility group box 2 significantly increased (p<0.01). Using an ex vivo proteomic approach in PBMC, we have identified circulating glucocorticoid-responsive proteins which may have potential as serum biomarkers of glucocorticoid activity.

Secretome Proteomic Approaches for Biomarker Discovery: An Update on Colorectal Cancer.

Searching for new cancer-related biomarkers is a key priority for the early detection of solid tumors, such as colorectal cancer (CRC), in clinically relevant biological fluids. The cell line and/or tumor tissue secretome represents a valuable resource for discovering novel protein markers secreted by cancer cells. The advantage of a secretome analysis is the reduction of the large dynamic range characterizing human plasma/serum, and the simultaneous enrichment of low abundance cancer-secreted proteins, thereby overcoming the technical limitations underlying the direct search in blood samples. In this review, we provided a comprehensive overview of recent studies on the CRC secretome for biomarker discovery, focusing both on methodological and technical aspects of secretome proteomic approaches and on biomarker-independent validation in CRC patient samples (blood and tissues). Secretome proteomics are mainly based on LC-MS/MS analyses for which secretome samples are either in-gel or in-solution trypsin-digested. Adequate numbers of biological and technical replicates are required to ensure high reproducibility and robustness of the secretome studies. Moreover, another major challenge is the accuracy of proteomic quantitative analysis performed by label-free or labeling methods. The analysis of differentially expressed proteins in the CRC secretome by using bioinformatic tools allowed the identification of potential biomarkers for early CRC detection. In this scenario, this review may help to follow-up the recent secretome studies in order to select promising circulating biomarkers to be validated in larger screenings, thereby contributing toward a complete translation in clinical practice.

Imbalance Between α1‐Antitrypsin and Neutrophil Elastase in Simple Steatosis Promotes Inflammation and Fibrosis Through Regulation of Adenosine a3 Receptor Signaling in Non‐alcoholic Steatohepatitis

Study objectivesNon‐alcoholic fatty liver disease (NAFLD) has been considered leading cause of developing liver failure. However, the underlying mechanisms for transition from benign steatosis to clinically significant non‐alcoholic steatohepatitis (NASH) and non‐alcoholic steatofibrosis (NASF) have not been fully understood.MethodIn mouse model, NAFLD was induced by fast food diet (FFD) with a high glucose‐fructose solution for 24 weeks. For secretome proteomics, we used primary hepatocytes (HPs) isolated from normal chow diet (NCD) or FFD‐fed liver. The biased agonist of A3 adenosine receptor (A3AR), FM101 was utilized for the study of A3AR signaling.ResultsIn secretome proteomics, we found α1‐antitrypsin (A1AT) was decreased 0.2‐fold in FFD hepatocytes compared with NCD hepatocytes. Consistently, serum levels of A1AT from mice and human patients with NAFLD were significantly decreased compared to normal group. Conversely, the mRNA and protein level of Neutrophil Elastase (NE) were increased in FFD‐fed liver compared to NCD‐fed liver. We next confirmed inflammatory role of NE in Kupffer cells (KCs) as various cytokines (CCL2, TNF‐α, IL‐1β, iNOS and IL‐6) were increased in KCs with treatment of NE. Of note, NE has been reported to exert proteolytic activity that can prevent activation of G‐protein‐coupled receptors (GPCRs). We observed similar proteolysis that A3AR, a member of the adenosine receptor group of GPCRs, was cleaved in hepatic stellate cells (HSCs) after treatment of NE, indicating that A1AT‐NE system may regulate the A3AR signaling. Interestingly, A3AR was predominantly expressed in KCs and HSCs, but not in HPs. In vitro activation of A3AR signaling with FM101 significantly inhibited activation of KCs and HSCs as demonstrated by inflammatory cytokines (TNF‐α, IL‐6 and IL‐1β), and profibrogenic genes (Col1a1, Timp1 Acta2) expression, respectively. Moreover, in vivo activation of A3AR with FM101, lowered liver weight, liver to body ratio, and serum level of ALT and cholesterol induced by FFD. Furthermore, FM101 effectively improved steatofibrosis as assessed by quantification of collagen deposition. Mechanistically, FM101 treatment strongly blocked mitochondrial oxidative phosphorylation. Using pMito‐Timer and Mt‐Keima system, we observed FM101‐induced mitochondria vulnerability as demonstrated by increased accumulation of damaged mitochondria and subsequence mitophagic degradation. Consequently, CCCP‐induced cell death in macrophage was further enhanced by FM101 treatment.ConclusionsThe imbalance between A1AT and NE in simple steatosis mediates the progression of NASH and NASF through proteolytic inactivation of A3AR in KCs and HSCs. Thus, A3AR agonism with FM101 may have therapeutic potentials for treatment of NAFLD.Support or Funding InformationNational Research Foundation of Korea (NRF) ‐ 2019R1A2C1090178FM101 ameliorates FFD‐induced steatohepatitis and stetofibrosisFigure 1Adenosine A3 receptor signaling contributes to mitochondria vulnerabilityFigure 2

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.

Combined Expression of Plasma Thrombospondin-2 and CA19-9 for Diagnosis of Pancreatic Cancer and Distal Cholangiocarcinoma: A Proteome Approach.

BackgroundMinimally invasive diagnostic biomarkers for patients with pancreatic ductal adenocarcinoma (PDAC) and distal cholangiocarcinoma (dCCA) are warranted to facilitate accurate diagnosis. This study identified diagnostic plasma proteins based on proteomics of tumor secretome.Materials and MethodsSecretome of tumor and normal tissue was collected after resection of PDAC and dCCA. Differentially expressed proteins were measured by mass spectrometry. Selected candidate biomarkers and carbohydrate antigen 19‐9 (CA19‐9) were validated by enzyme‐linked immunosorbent assay in plasma from patients with PDAC (n = 82), dCCA (n = 29), benign disease (BD; n = 30), and healthy donors (HDs; n = 50). Areas under the curve (AUCs) of receiver operator characteristic curves were calculated to determine the discriminative power.ResultsIn tumor secretome, 696 discriminatory proteins were identified, including 21 candidate biomarkers. Thrombospondin‐2 (THBS2) emerged as promising biomarker. Abundance of THBS2 in plasma from patients with cancer was significantly higher compared to HDs (p < .001, AUC = 0.844). Combined expression of THBS2 and CA19‐9 yielded the optimal discriminatory capacity (AUC = 0.952), similarly for early‐ and late‐stage disease (AUC = 0.971 and AUC = 0.911). Remarkably, this combination demonstrated a power similar to CA19‐9 to discriminate cancer from BD (AUC = 0.764), and THBS2 provided an additive value in patients with high expression levels of bilirubin.ConclusionOur proteome approach identified a promising set of candidate biomarkers. The combined plasma expression of THBS2/CA19‐9 is able to accurately distinguish patients with PDAC or dCCA from HD and BD.Implications for PracticeThe combined plasma expression of thrombospondin‐2 and carbohydrate antigen 19‐9 is able to accurately diagnose patients with pancreatic cancer and distal cholangiocarcinoma. This will facilitate minimally invasive diagnosis for these patients by distinguishing them from healthy individuals and benign diseases.

Bioinformatics Tools and Workflow to Select Blood Biomarkers for Early Cancer Diagnosis: An Application to Pancreatic Cancer.

Secretome proteomics for the discovery of cancer biomarkers holds great potential to improve early cancer diagnosis. A knowledge-based approach relying on mechanistic criteria related to the type of cancer should help to identify candidates from available "omics" information. With the aim of accelerating the discovery process for novel biomarkers, a set of tools is developed and made available via a Galaxy-based instance to assist end-users biologists. These implemented tools proceed by a step-by-step strategy to mine transcriptomics and proteomics databases for information relating to tissue specificity, allow the selection of proteins that are part of the secretome, and combine this information with proteomics datasets to rank the most promising candidate biomarkers for early cancer diagnosis. Using pancreatic cancer as a case study, this strategy produces a list of 24 candidate biomarkers suitable for experimental assessment by MS-based proteomics. Among these proteins, three (SYCN, REG1B, and PRSS2) were previously reported as circulating candidate biomarkers of pancreatic cancer. Here, further refinement of this list allows to prioritize 14 candidate biomarkers along with their associated proteotypic peptides for further investigation, using targeted MS-based proteomics. The bioinformatics tools and the workflow implementing this strategy for the selection of candidate biomarkers are freely accessible at http://www.proteore.org.

Abstract 2552: Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses

Abstract Cancer immunotherapies have shown remarkable clinical activity across several tumor types but responses have been modest in breast cancers. This is attributed to low levels of neo-antigens in breast tumors and immunosuppressive nature of mammary tumor microenvironment. Here we investigated if we can counteract these obstacles to an effective immunotherapy by inducing senescence in breast cancer cells. Senescence is one of the physiological stress response programs characterized by loss of proliferative potential and secretion of a variety of pro-inflammatory markers which promotes identification and removal of senescent cells by the immune cells. Here we hypothesized that senescence will stimulate anti-tumor immune response by establishing immune-permissive microenvironment. We utilized immunocompetent and humanized PDX-bearing mice to investigate senescence-induced modulation of tumor secretome proteome, transcriptome and immune cell recruitment and activity. An FDA-approved small molecule CDK4/6 inhibitor was used to induce senescence. We detected induction of multiple markers of senescence after CDK4/6 inhibitor treatment of cells and tumors. Senescent cells and tumors had increased secretion of chemokines CCL4, CCL5, CXCL9, CXCL10 and CXCL11 which facilitated T cell homing into the tumor. This was a result of their transcriptional induction by NF-κB. Consequently, we observed the dynamic enrichment of tumor immune microenvironment with T cells in tumors treated with CDK4/6 inhibitor. Furthermore, the recruitment of adoptively transferred ex-vivo activated anti-tumor T cells was significantly enhanced in tumors that were treated with CDK4/6 inhibitor. Finally, CDK4/6 inhibitor-treated tumors became responsive to T cell-activating immunotherapy with antibody agonists of T cell co-stimulatory receptors OX40 and 4-1BB. In summary, our findings suggest that CDK4/6 inhibition can facilitate T cell recruitment into the mammary tumors which can lead to an improved immunotherapy response. This pre-clinical study provides rationale for clinical development of CDK4/6 inhibitors and T cell agonists and uncovers the potential of senescence-inducing approach for sensitizing tumors to immune therapy. Citation Format: Ashlyn Blevins, Stacey Mont, Hunter Lawrence, Nabil Saleh, Sheau-Chiann Chen, Ann Richmond, Anna Vilgelm. Senescence induced by CDK4/6 inhibition facilitates anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2552.

Hypoxia-Induced Changes in the Fibroblast Secretome, Exosome, and Whole-Cell Proteome Using Cultured, Cardiac-Derived Cells Isolated from Neonatal Mice.

Cardiac fibroblasts (CFs) represent a major subpopulation of cells in the developing and adult heart. Cardiomyocyte (CM) and CF intercellular communication occurs through paracrine interactions and modulate myocyte development and stress response. Detailed proteomic analysis of the CF secretome in normal and stressed conditions may offer insights into the role of CF in heart development and disease. Primary neonatal mouse CFs were isolated and cultured for 24 h in 21% (normoxic) or 2% (hypoxic) O2. Conditioned medium was separated to obtain exosomes (EXO) and EXO-depleted secretome fractions. Multidimensional protein identification technology was performed on secreted fractions. Whole cell lysate data were also generated to provide subcellular context to the secretome. Proteomic analysis identified 6163 unique proteins in total. Statistical (QSpec) analysis identified 494 proteins differentially expressed between fractions and oxygen conditions. Gene Ontology enrichment analysis revealed hypoxic conditions selectively increase expression of proteins with extracellular matrix and signaling annotations. Finally, we subjected CM pretreated with CF secreted factors to hypoxia/reoxygenation. Viability assays suggested altered viability due to CF-derived factors. CF secretome proteomics revealed differential expression based on mode of secretion and oxygen levels in vitro.

RETRACTED: Exosomes promote restoration after an experimental animal model of intracerebral hemorrhage

Exosomes are gaining importance because they show great promise in therapeutic applications for several diseases. Particularly in stroke, exosomes derived from mesenchymal stem cell (MSC) therapy work as paracrine effectors responsible for promoting neurovascular remodeling and functional recovery. Adult male rats were subjected to intracerebral hemorrhage (ICH) by intrastriatal injection of collagenase type IV; 24 h after surgery, MSC-derived exosomes were administered through the tail vein. The rats were euthanized at 7 or 28 days after treatment. Functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers, biodistribution of exosomes and secretome proteomics were analyzed. DiI-labeled exosomes were found in the brains of the ICH-treated group and in the liver, lung and spleen. Animals receiving treatment with exosomes showed significantly better results in terms of functional recovery, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group 28 days after stroke. Proteomics analysis of the exosomes identified more than 2000 proteins that could be implicated in brain repair function. In conclusion, white matter integrity was partly restored by exosome administration mediated by molecular repair factors. Exosomes as a treatment could be a heterogeneous process by nature and presents many factors that can influence brain plasticity in an adaptable and versatile manner.

Secretome proteomics reveals candidate non-invasive biomarkers of BRCA1 deficiency in breast cancer.

Breast cancer arising in female BRCA1 mutation carriers is characterized by an aggressive phenotype and early age of onset. We performed tandem mass spectrometry-based proteomics of secretomes and exosome-like extracellular vesicles from BRCA1-deficient and BRCA1-proficient murine breast tumor models to identify extracellular protein biomarkers, which can be used as an adjunct to current diagnostic modalities in patients with BRCA1-deficient breast cancer. We identified 2,107 proteins, of which 215 were highly enriched in the BRCA1-deficient secretome. We demonstrated that BRCA1-deficient secretome proteins could cluster most human BRCA1- and BRCA2-related breast carcinomas at the transcriptome level. Topoisomerase I (TOP1) and P-cadherin (CDH3) expression was investigated by immunohistochemistry on tissue microarrays of a large panel of 253 human breast carcinomas with and without BRCA1/2 mutations. We showed that expression of TOP1 and CDH3 was significantly increased in human BRCA1-related breast carcinomas relative to sporadic cases (p = 0.002 and p < 0.001, respectively). Multiple logistic regression showed that TOP1 (adjusted odds ratio [OR] 3.75; 95% confidence interval [95% CI], 1.85 - 7.71, p < 0.001) as well as CDH3 positivity (adjusted OR 2.45; 95% CI, 1.08 - 5.49, p = 0.032) were associated with BRCA1/2-related breast carcinomas after adjustment for triple-negative phenotype and age. In conclusion, proteome profiling of secretome using murine breast tumor models is a powerful strategy to identify non-invasive candidate biomarkers of BRCA1-deficient breast cancer. We demonstrate that TOP1 and CDH3 are closely associated to BRCA1-deficient breast cancer. These data merit further investigation for early detection of tumors arising in BRCA1 mutation carriers.

Tumour-processed osteopontin and lactadherin drive the protumorigenic reprogramming of microglia and glioma progression.

Tumour tissue is infiltrated by myeloid cells that are reprogrammed into alternatively activated/regenerative (M2) macrophages. The contribution of major signalling pathways and their modulators/targets involved in the macrophage reprogramming is poorly known. Glioblastoma (malignant brain tumour) attracts and reprograms brain-resident microglia and peripheral macrophages into cells that increase invasion, angiogenesis and suppress antitumour immunity. Using a 'function-first' approach and glioma secretome proteomics we identified osteopontin and lactadherin as proteins that cooperatively activate amoeboid transformation, phagocytosis and motility of primary microglia cultures via integrins and FAK-Akt (focal adhesion kinase-Akt) signalling. A synthetic peptide interfering with integrin ligands blocks glioma-microglia communication, functional activation and M2 gene expression. We found that osteopontin/secreted phosphoprotein 1 (Spp1) produced by non-transformed cells acts as a proinflammatory factor inducing inflammatory signalling and M1 genes, and counteracts the action of lactadherin. Using constructs encoding functional mutants of osteopontin, we demonstrated sequential processing of Spp1 by thrombin and matrix metalloproteinase-3 and/or -7 (MMP-3 and/or -7) in glioma cells, which generates a microglia-activating form devoid of the inflammatory activity, while retaining the M2 reprogramming potential. A similar form of osteopontin is secreted by human glioma cells but not normal human astrocytes. Knockdown of osteopontin or lactadherin in glioma cells reduces intracranial glioma growth, blocks amoeboid transformation of myeloid cells and affects M2 reprogramming of microglia/macrophages. Our findings demonstrate how glioma cells misuse macrophage-activating signals and redesign primarily proinflammatory signals towards their advantage to induce M2 reprogramming of tumour-infiltrating brain macrophages.

Exosomes Secreted by Apoptosis-Resistant Acute Myeloid Leukemia (AML) Blasts Harbor Regulatory Network Proteins Potentially Involved in Antagonism of Apoptosis

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividualex vivoapoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p= 0.0067 andp= 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p= 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.