Protein Secretion Profile Research Articles

Temporal quantitative proteome analysis reveals dynamic change of cellular and secreted protein profiles of Clostridium cellulovorans depending on carbon sources

An anaerobic bacterium Clostridium cellulovorans secretes a large cell‐surface enzyme complex called cellulosome and this can degrade and metabolize cellulose, hemicellulose, and pectin which constitute plant cell wall. Furthermore, C. cellulovorans optimizes profiles of cellulosomal enzymes and non‐cellulosomal enzymes, which are secreted to media, depending on carbon sources. [1] Under the degradation of each carbon source, dynamical changes of composition of metabolic proteins occurred. [2] C. cellulovorans recognizes carbon sources in media and optimizes cell surface, secreted, and metabolic proteins to adapt to environment.It is very interesting to reveal a dynamic adaptation mechanism of cellular and secreted protein profiles depending on carbon sources. In addition, temporal microbial profiling of cellular and secreted protein derived from the one‐pot culture has not been reported. In this research, we cultured C. cellulovorans in 5 carbon sources and took cellular and secreted samples temporally. After that, quantitative proteome analysis using tandem mass tag was carried out. From this analysis, we attempted to clarify temporal dynamic changes of protein profiles depending on carbon sources and culture times.After the cultivation and sampling of each time point in glucose, cellulose, xylan, pectin, and galactomannan, the cellular proteins were extracted and the secreted proteins were concentrated. Each protein was alkylated after reduction, and digested by trypsin. Peptides were labeled with the stable isotope label and subjected to the LC‐MS/MS system equipped with unique super‐long monolithic silica capillary column. [2]We could totally identify about 1500 proteins of C. cellulovorans in one‐shot proteome analysis, and these profiles were significantly dependent on each substrate and time point. Obtained data were analyzed statistically and changing proteins depending on substrates and time points was significantly identified. Focusing on central metabolic enzymes, we could clarify dynamic changes of these enzymes with time‐ and substrate‐depending manners. Cellulosomal and non‐cellulosomal proteins derived from each fraction are also dynamically changed as carbon sources were degraded. From these results, we considered adaptation and degradation mechanisms of C. cellulovorans with various carbon sources. [3]

A Bioprocessed Polysaccharide from Lentinus edodes Mycelia Cultures with Turmeric Protects Chicks from a Lethal Challenge of Salmonella Gallinarum

Our previous studies demonstrated that a bioprocessed polysaccharide (BPP) isolated from Lentinus edodes mushroom mycelia cultures supplemented with black rice bran can protect mice against Salmonella lipopolysaccharide-induced endotoxemia and reduce the mortality from Salmonella Typhimurium infection through upregulated T-helper 1 immunity. Here, we report that a BPP from L. edodes mushroom mycelia liquid cultures supplemented with turmeric (referred to as BPP-turmeric) alters chicken macrophage responses against avian-adapted Salmonella Gallinarum and protects chicks against a lethal challenge from Salmonella Gallinarum. In vitro analyses revealed that the water extract of BPP-turmeric (i) changed the protein expression or secretion profile of Salmonella Gallinarum, although it was not bactericidal, (ii) reduced the phagocytic activity of the chicken-derived macrophage cell line HD-11 when infected with Salmonella Gallinarum, and (iii) significantly activated the transcription expression of interleukin (IL)-1β, IL-10, tumor necrosis factor α, and inducible nitric oxide synthase in response to various Salmonella infections, whereas it repressed that of IL-4, IL-6, interferon-β, and interferon-γ. We also found that BPP-turmeric (0.1 g/kg of feed) as a feed additive provided significant protection to 1-day-old chicks infected with a lethal dose of Salmonella Gallinarum. Collectively, these results imply that BPP-turmeric contains biologically active component(s) that protect chicks against Salmonella Gallinarum infection, possibly by regulating macrophage immune responses. Further studies are needed to evaluate the potential efficacy of BPP-turmeric as a livestock feed additive for the preharvest control of fowl typhoid or foodborne salmonellosis.

Profile of Endometrial Secretory Proteins in Repeat Breeder Jersey Crossbred Cows

Profile of Endometrial Secretory Proteins in Repeat Breeder Jersey Crossbred Cows

SILAC-based quantitative proteomic analysis of secretome of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV-infected Marc-145 cells compared with mock-infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle-mediated release of exosomal proteins, including different danger-associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV-infected Marc-145 cells and provides valuable insight into the host response to PRRSV infection.

The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress.

BackgroundNTPases (also NTPDases) are enzymes with apyrase activity. They are widely distributed among eukaryotes, and also among members of the family Sarcocystidae. In Toxoplasma gondii, the TgNTPase accumulates in the dense granules, and has been commonly associated with the strain virulence. In the closely related Neospora caninum, the NcNTPase lacks nucleoside diphosphate hydrolase activity and appears to be more abundant in virulent isolates, indicating that it may contribute to the pathogenicity of neosporosis. However, so far no additional information on NcNTPase has been provided.MethodsHerein, the NcNTPase coding sequences were analysed by different in silico and de novo sequencing approaches. A comparative analysis of NcNTPase and NcGRA7 in terms of protein dynamics, secretion, phosphorylation, and mRNA expression profiles during the tachyzoite lytic cycle was also carried out. Moreover, NcNTPase immunolocalization was analysed by confocal microscopy techniques over a set number of time-points.ResultsWe describe the presence of three different loci containing three copies of the NcNTPase within the Nc-Liv genome, and report the existence of up to four different NcNTPase alleles in Nc-Liv. We also provide evidence for the occurrence of diverse protein species of the NcNTPase by two-dimensional gel electrophoresis. Both NcNTPase and NcGRA7 were similarly up-regulated and secreted during the egress and/or early invasion phases, and were phosphorylated. However, its secretion was not affected by the addition of calcium modulators such as A23187 and ethanol. NcNTPase and NcGRA7 localized in dense granules and parasitophorous vacuole membrane throughout the lytic cycle, although differed in their inmunolocalization during early invasion and egress.ConclusionsThe present study reveals the complexity of the NcNTPase loci in N. caninum. We hypothesize that the expression of different isoforms of the NcNTPase protein could contribute to parasite virulence. Our findings showed regulation of expression, secretion and phosphorylation of NcNTPase suggesting a potential role for progression through the tachyzoites lytic cycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1620-4) contains supplementary material, which is available to authorized users.

Proteomic Profile of Unstable Atheroma Plaque: Increased Neutrophil Defensin 1, Clusterin, and Apolipoprotein E Levels in Carotid Secretome.

Because of the clinical significance of carotid atherosclerosis, the search for novel biomarkers has become a priority. The aim of the present study was to compare the protein secretion profile of the carotid atherosclerotic plaque (CAP, n = 12) and nonatherosclerotic mammary artery (MA, n = 10) secretomes. We used a nontargeted proteomic approach that incorporated tandem immunoaffinity depletion, iTRAQ labeling, and nanoflow liquid chromatography coupled to high-resolution mass spectrometry. In total, 162 proteins were quantified, of which 25 showed statistically significant differences in secretome levels between carotid atherosclerotic plaque and nondiseased mammary artery. We found increased levels of neutrophil defensin 1, apolipoprotein E, clusterin, and zinc-alpha-2-glycoprotein in CAP secretomes. Results were validated by ELISA assays. Also, differentially secreted proteins are involved in pathways such as focal adhesion and leukocyte transendothelial migration. In conclusion, this study provides a subset of identified proteins that are differently expressed in secretomes of clinical significance.

Provisional Matrix Citrullination Contributes to Enhanced Fibroblast Migration

Activated fibroblasts—characterized by various altered behaviors including invasiveness, altered protein secretion profiles, and resistance to apoptosis—contribute to the development and exacerbation of numerous chronic human inflammatory diseases including cancer, rheumatoid arthritis, and interstitial lung disease. Citrullination, a post-translational protein modification known to occur extensively in inflammatory environments, alters the provisional extracellular matrix (ECM) and key arginine-containing cell-binding sites therein. We hypothesize that citrullination alters fibroblast interactions with provisional ECM such that adhesion is reduced and related downstream functions such as motility and contraction are also altered. Results have thus far indicated that provisional matrix citrullination does contribute, solely or in part, to several characteristics of activated fibroblasts. Adhesion assays, whereby human foreskin fibroblasts (HFFs) were allowed to attach to modified or normal provisional ECM and subsequently challenged with shear forces, suggest that citrullination significantly decreases fibroblast attachment and spreading. Blocking experiments have shown that these effects appear to be mediated, in part, by both RGD and αvβ3 integrin interactions. Despite this decreased adhesion, in vitro random migration and wound healing assays have paradoxically demonstrated the ability of citrullinated provisional ECM to significantly enhance fibroblast migration rates, indicating that adhesion differences alone do not fully explain differences in citrullination-mediated cell motility. This research will ultimately provide insight for understanding a fundamental pathway linking citrullination of provisional ECM proteins with fibroblast activation. It therefore constitutes an important step in the development of novel treatments to both prevent and ameliorate a panoply of human diseases, many of which currently have inadequate or nonexistent therapeutic solutions.

Proteomic analysis reveals differentially secreted proteins in the urine from patients with clear cell renal cell carcinoma

ObjectiveThe aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass spectrometry–based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. Methods and materialsUrine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry–based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MSE (1D LC-MSE), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. ResultsAll the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MSE quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. ConclusionsThe proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MSE and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.

Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell-derived adipocytes.

One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.

Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona

Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells’ protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

Quantitative Proteomic Analysis of Burkholderia pseudomallei Bsa Type III Secretion System Effectors Using Hypersecreting Mutants

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. We constructed a mutant lacking bsaP, a homolog of the T3SS “gatekeeper” family of proteins that exert control over the timing and magnitude of effector protein secretion. Mutants lacking BsaP, or the T3SS translocon protein BipD, were observed to hypersecrete the known Bsa effector protein BopE, providing evidence of their role in post-translational control of the Bsa T3SS and representing key reagents for the identification of its secreted substrates. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hypersecreting mutants of B. pseudomallei with the isogenic parent strain and a bsaZ mutant incapable of effector protein secretion. Our study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei.

Toxicoproteomic analysis of pulmonary carbon nanotube exposure using LC-MS/MS.

Toxicoproteomics is a developing field that utilizes global proteomic methodologies to investigate the physiological response as a result of adverse toxicant exposure. The aim of this study was to compare the protein secretion profile in lung bronchoalveolar lavage fluid (BALF) from mice exposed to non-functionalized multi-walled carbon nanotubes (U-MWCNTs) or MWCNTs functionalized by nanoscale Al2O3 coatings (A-MWCNT) formed using atomic layer deposition (ALD). Proteins were identified using liquid chromatography tandem mass spectrometry (LC-MS/MS), and quantified using a combination of two label-free proteomic methods: spectral counting and MS1 peak area analysis. On average 465 protein groups were identified per sample and proteins were first screened using spectral counting and the Fisher's exact test to determine differentially regulated species. Significant proteins by Fisher's exact test (p<0.05) were then verified by integrating the intensity under the extracted ion chromatogram from a single unique peptide for each protein across all runs. A two sample t-test based on integrated peak intensities discovered differences in 27 proteins for control versus U-MWCNT, 13 proteins for control versus A-MWCNT, and 2 proteins for U-MWCNT versus A-MWCNT. Finally, an in-vitro binding experiment was performed yielding 4 common proteins statistically different (p<0.05) for both the in-vitro and in-vivo study. Several of the proteins found to be significantly different between exposed and control groups are known to play a key role in inflammatory and immune response. A comparison between the in-vitro and in-vivo CNT exposure emphasized a true biological response to CNT exposure.

Proteomic Comparison of the Secreted Factors of Mesenchymal Stem Cells from Bone Marrow, Adipose Tissue and Dental Pulp

Mesenchymal stem cells (MSCs) have therapeutic potential in various diseases, including myocardial infarction. Recently, the importance of the therapeutic effects of secreted factors from MSCs is increasing, but their identification has not progressed. In this study, we uncover the secreted protein profiles of MSCs derived from bone marrow, adipose tissue and dental pulp. Shotgun proteome analysis of conditioned MSC media by mass spectrometry identified 1533 proteins totally and 124 secreted proteins commonly produced among all three MSCs. The commonly secreted factors include already well-known factors whose functions are linked to MSCs’ biological effects, such as CTGF, SERPINE1, TGFB1, DKK3 and MYDGF, and also include newly identified factors whose roles are not well investigated, for example AIMP1, CLEC11A, GAS6, HDGF, INHBA, and PCSK5. Computational biological pathway analysis revealed that these common factors strongly relate to tissue regeneration pathways such as angiogenesis, migration, and inflammatory response. Further analysis showed enrichment of ossification, sprouting, and organ survival factors, suggesting connection to the functions closely related to MSCs’ therapeutic effects. This list of commonly secreted proteins could provide a reliable resource of biological factors which explain various effects of MSCs and would be useful for identifying new therapeutic factors produced from MSCs

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Different culture conditions modulate the immunological properties of adipose stem cells.

The potential of human adipose stem cells (ASCs) for regenerative medicine has received recognition owing to their ease of isolation and their multilineage differentiation capacity. Additionally, low immunogenicity and immunosuppressive properties make them a relevant cell source when considering immunomodulation therapies and allogeneic stem cell treatments. In the current study, immunogenicity and immunosuppression of ASCs were determined through mixed lymphocyte reactions. The immunogenic response was analyzed after cell isolation and expansion in fetal bovine serum (FBS), human serum (HS)-supplemented medium, and xeno-free and serum-free (XF/SF) conditions. Additionally, the immunophenotype and the secretion of CXC chemokine ligand 8 (CXCL8), CXCL9, CXCL10, C-C chemokine ligand 2 (CCL2), CCL5, interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor-α, interferon-γ, transforming growth factor-β1, indoleamine 2,3-deoxygenase, Galectin-1, and Galectin-3 were analyzed. The results showed that ASCs were weakly immunogenic when expanded in any of the three conditions. The significantly strongest suppression was observed with cells expanded in FBS conditions, whereas higher ASC numbers were required to display suppression in HS or XF/SF conditions. In addition, statistically significant differences in protein secretion were observed between direct versus indirect cocultures and between different culture conditions. The characteristic immunophenotype of ASCs was maintained in all conditions. However, in XF/SF conditions, a significantly lower expression of CD54 (intercellular adhesion molecule 1) and a higher expression of CD45 (lymphocyte common antigen) was observed at a low passage number. Although culture conditions have an effect on the immunogenicity, immunosuppression, and protein secretion profile of ASCs, our findings demonstrated that ASCs have low immunogenicity and promising immunosuppressive potential whether cultured in FBS, HS, or XF/SF conditions.

Real-Time Quantification of Proteins Secreted by Artificial Connective Tissue Made from Uni- Or Multidirectional Collagen I Scaffolds and Oral Mucosa Fibroblasts

Previously, we found that oral autologous artificial connective tissue (AACT) had a different protein secretion profile to that of clot-embedded AACT. Other oral mucosa substitutes, having different cell types and scaffolds, had dissimilar secretion profiles of proteins (including that for AACT) that influence healing outcome; thus, to ascertain the profiles of factors secreted by artificial tissue and whether they are influenced by their microstructure might help in understanding their bioactivity. An important component of tissue microstructure is the fiber orientation of the scaffold used for manufacturing it. This work developed a surface plasmon resonance (SPR) methodology to quantify factors secreted by oral artificial connective tissue (ACT) in culture medium, and a method to manufacture unidirectional laminar collagen I scaffolds. The SPR methodology was used for assessing differences in the protein secretion profile of ACT made with collagen scaffolds having different fiber orientation (unidirectional vs multidirectional). Oral fibroblasts seeded onto unidirectional scaffolds increased the secretion of six factors involved in modulating healing compared to those seeded onto multidirectional scaffolds. Histological analysis of uni- and multidirectional ACT showed that cells differ in their alignment and morphology. This SPR-methodology led to nanoscale detection of paracrine factors and might be useful to study biomarkers of three-dimensional cell growth, cell differentiation, and wound-healing progression.

Assessment of Heteroporus biennis secretion extracts for decolorization of textile dyes

In the present study, the dye-decolorizing properties of soluble extracts from the indigenous fungus Heteroporus biennis IOC4581 were determined. Decolorizing activity was associated with the secretion of a 45-kDa laccase. Laccase expression was extremely sensitive to carbon and nitrogen contents in culture media. Supplementation with metal ions and aromatic compounds resulted in extremely variable protein secretion profiles, although enzyme yields were not improved. Kinetic parameters of the soluble crude extract were promising, destaining seven out of nine textile dyes without the need of redox mediators. The extract was insensitive to Ca2+, Mg2+, Mn2+, and Zn2+ ions, and catalysis was enhanced in the presence of Cu2+ and Al3+ ions. The soluble fungal extract retained its maximal decolorizing activity at a wide range of pH (2.0–9.0) and temperature (4–50 °C), indicating potential application to dye biodegradation.

Stroke Literature Synopses : Basic Sciences

There remains some plasticity even in adult brain, and after stroke, the brain tries to remodel damaged neuronal circuits. Neuronal precursor cells (NPCs) may participate in this process, but how NPCs move to lesion areas from subventricular zone (SVZ) remains to be fully understood. Three recent studies provide new fundamental mechanisms for neural migration in the central nervous system. Itoh et al (Scratch regulates neuronal migration onset via an epithelial-mesenchymal transition-like mechanism. Nature Neurosci . 2013;16:416–425) proposed a novel mechanism for the conversion of neuroepithelial cells to migrating neurons or intermediate NPCs. During development, NPCs in the SVZ form a pseudostratified epithelial structure, and once they commit to becoming neurons (or intermediate NPCs), they delaminate and start migration. To reveal the underlying regulatory mechanisms, the authors focused on roles of Scratch1 and Scratch2, which are members of the Snail superfamily of transcription factors. They knocked down Scratch1 and Scratch2 in the neocortical NPCs during neocortical development by …

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction.

Secreted proteins including cytokines, chemokines, and growth factors represent important functional regulators mediating a range of cellular behavior and cell–cell paracrine/autocrine signaling, e.g., in the immunological system (Rothenberg, 2007), tumor microenvironment (Hanahan and Weinberg, 2011), or stem cell niche (Gnecchi etal., 2008). Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically identical cell population can give rise to diverse phenotypic differences (Niepel etal., 2009). Non-genetic heterogeneity is also emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies (Cohen etal., 2008; Gascoigne and Taylor, 2008), but can hardly assessed using conventional approaches that do not examine cellular phenotype at the functional level. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

Differential secretome analysis of cancer‐associated fibroblasts and bone marrow‐derived precursors to identify microenvironmental regulators of colon cancer progression

The identification of cancer-associated fibroblast (CAF)-derived proteins that mediate interactions between the tumor stroma and cancer cells is a crucial step toward the discovery of new molecular targets for therapy or molecular signatures that improve tumor classification and predict clinical outcome. CAF are α-smooth muscle actin positive, representing a myofibroblast phenotype that may differentiate from multiple precursor cells, including bone marrow-derived mesenchymal stem cells (MSC). Transforming growth factor-β1 (TGF-β1) is a crucial inducer of α-smooth muscle actin positive CAFs. In this study, we aimed to identify CAF-derived regulators of colon cancer progression by performing a high-throughput differential secretome profiling between CAF compared to noncancer-activated bone marrow-derived MSC. In addition, we explored the effect of TGF-β1 on the secretion of proteins by bone marrow-derived MSC in comparison with the protein secretion profile of CAF. TGF-β1 induced de novo secretion of 84 proteins in MSC, of which 16 proteins, including stromal-derived factor-1α and Rantes, were also present in CAF secretome. Immunohistochemistry further validated the expression of selected candidates such as tenascin C, fibronectin ED-A domain and stromal-derived factor-1 in clinical colon cancer specimens. In conclusion, this differential secretome approach enabled us to identify a series of candidate biomarkers for colon cancer that are associated with a CAF-specific phenotype.