Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma.

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.

Tumor-derived exosomes and microvesicles in head and neck cancer: implications for tumor biology and biomarker discovery.

Exosomes and microvesicles (MVs) are nanometer-sized, membranous vesicles secreted from many cell types into their surrounding extracellular space and into body fluids. These two classes of extracellular vesicles are regarded as a novel mechanism through which cancer cells, including virally infected cancer cells, regulate their micro-environment via the horizontal transfer of bioactive molecules: proteins, lipids, and nucleic acids (DNA, mRNA, micro-RNAs; oncogenic cargo hence often referred to as oncosomes). In head and neck cancer (HNC), exosomes and MVs have been described in Epstein Barr Virus (EBV)-associated nasopharyngeal cancer (NPC), as well as being positively correlated with oral squamous cell carcinoma (OSCC) progression. It has therefore been suggested that HNC-derived vesicles could represent a useful source for biomarker discovery, enriched in tumor antigens and cargo; hence fundamentally important for cancer progression. This current review offers an overall perspective on the roles of exosomes and MVs in HNC biology, focusing on EBV-associated NPC and OSCC. We also highlight the importance of saliva as a proximal and easily accessible bio-fluid for HNC detection, and propose that salivary vesicles might serve as an alternative model in the discovery of novel HNC biomarkers.

In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine.

Expressed prostatic secretions (EPS) are proximal fluids of the prostate that are increasingly being utilized as a clinical source for diagnostic and prognostic assays for prostate cancer (PCa). These fluids contain an abundant amount of microvesicles reflecting the secretory function of the prostate gland, and their protein composition remains poorly defined in relation to PCa. Using expressed prostatic secretions in urine (EPS-urine), exosome preparations were characterized by a shotgun proteomics procedure. In pooled EPS-urine exosome samples, ~900 proteins were detected. Many of these have not been previously observed in the soluble proteome of EPS generated by our labs or other related exosome proteomes. We performed systematic comparisons of our data against previously published, prostate-related proteomes, and global annotation analyses to highlight functional processes within the proteome of EPS-urine derived exosomes. The acquired proteomic data have been deposited to the Tranche repository and will lay the foundation for more extensive investigations of PCa derived exosomes in the context of biomarker discovery and cancer biology.

Interleukin-22 and interleukin-22-producing NKp44+ natural killer cells in subclinical gut inflammation in ankylosing spondylitis.

OBJECTIVE: The intestinal inflammation observed in patients with ankylosing spondylitis (AS) is characterized by an overexpression of interleukin-23 (IL-23). IL-23 is known to regulate IL-22 production through lamina propria NKp44+ natural killer (NK) cells, which are thought to be involved in protective mucosal mechanisms. This study was undertaken to evaluate the frequency of NKp44+ NK cells and the expression of IL-22 in the ileum of AS patients. METHODS: Tissue NKp44+ NK cells, NKp46+ NK cells, and IL-22-producing cells were analyzed by flow cytometry. Quantitative gene expression analysis of IL-22, IL-23, IL-17, STAT-3, and mucin 1 (MUC-1) was performed by reverse transcriptase-polymerase chain reaction on ileal samples from 15 patients with AS, 15 patients with Crohn's disease (CD), and 15 healthy controls. NKp44, pSTAT-3, and IL-22 expression was analyzed by immunohistochemistry. RESULTS: The frequency of NKp44+ but not NKp46+ NK cells was increased in the inflamed ileum of AS patients compared to CD patients and controls. The frequency of NKp46+ NK cells was significantly increased only in CD patients. Among CD4+ lymphocytes and NKp44+ NK cell subsets, the latter were the major source of IL-22 on lamina propria mononuclear cells from AS patients. Significant up-regulation of IL-22, IL-23p19, MUC-1, and STAT-3 transcripts in the terminal ileum of patients with AS was observed. Immunohistochemical analysis confirmed the increased IL-22 and pSTAT-3 expression in inflamed mucosa from AS and CD patients. CONCLUSION: Our findings indicate that overexpression of IL-22, together with an increased number of IL-22-producing NKp44+ NK cells, occurs in the gut of AS patients, where it appears to play a tissue-protective role.

Identification of prostate-enriched proteins by in-depth proteomic analyses of expressed prostatic secretions in urine.

Urinary expressed prostatic secretion or "EPS-urine" is proximal tissue fluid that is collected after a digital rectal exam (DRE). EPS-urine is a rich source of prostate-derived proteins that can be used for biomarker discovery for prostate cancer (PCa) and other prostatic diseases. We previously conducted a comprehensive proteome analysis of direct expressed prostatic secretions (EPS). In the current study, we defined the proteome of EPS-urine employing Multidimensional Protein Identification Technology (MudPIT) and providing a comprehensive catalogue of this body fluid for future biomarker studies. We identified 1022 unique proteins in a heterogeneous cohort of 11 EPS-urines derived from biopsy negative noncancer diagnoses with some benign prostatic diseases (BPH) and low-grade PCa, representative of secreted prostate and immune system-derived proteins in a urine background. We further applied MudPIT-based proteomics to generate and compare the differential proteome from a subset of pooled urines (pre-DRE) and EPS-urines (post-DRE) from noncancer and PCa patients. The direct proteomic comparison of these highly controlled patient sample pools enabled us to define a list of prostate-enriched proteins detectable in EPS-urine and distinguishable from a complex urine protein background. A combinatorial analysis of both proteomics data sets and systematic integration with publicly available proteomics data of related body fluids, human tissue transcriptomic data, and immunohistochemistry images from the Human Protein Atlas database allowed us to demarcate a robust panel of 49 prostate-derived proteins in EPS-urine. Finally, we validated the expression of seven of these proteins using Western blotting, supporting the likelihood that they originate from the prostate. The definition of these prostatic proteins in EPS-urine samples provides a reference for future investigations for prostatic-disease biomarker studies.

Expression of interleukin-32 in the inflamed arteries of patients with giant cell arteritis.

OBJECTIVE: Giant cell (temporal) arteritis (GCA) is a vasculitis that mainly affects the large and medium arteries, especially the branches of the proximal aorta. Interleukin-32 (IL-32) is a recently described Th1 proinflammatory cytokine, and is mainly induced by interferon-γ (IFNγ), IL-1ß, and tumor necrosis factor α (TNFα). This study was undertaken to investigate the expression and tissue distribution of IL-32 in artery biopsy specimens from patients with GCA. METHODS: Quantitative gene expression analysis of IL-32, IL-1ß, TNFα, IFNγ, IL-6, and IL-27 was performed in artery biopsy specimens obtained from 18 patients with GCA and 15 controls. Immunohistochemistry analysis was performed to evaluate IL-32 tissue distribution and identify IL-32-producing cells. Circulating Th1 lymphocytes were evaluated by flow cytometry. RESULTS: We demonstrated a strong and significant up-regulation of IL-32 at both the messenger RNA and protein levels in the artery biopsy samples from patients with GCA. IL-32 was abundantly expressed by vascular smooth muscle cells of inflamed arteries and neovessels within inflammatory infiltrates. IL-32 expression strongly correlated with the intensity of the systemic inflammatory response. IL-32 overexpression was accompanied by strong overexpression of Th1 cytokines, such as IFNγ and IL-27p28, in inflamed arteries from GCA patients. The Th1 lymphocyte population was also expanded among peripheral blood mononuclear cells from GCA patients and produced higher amounts of IL-32 compared to controls. CONCLUSION: Our findings indicate that overexpression of IL-32 together with a clear Th1 response immunologically characterizes the inflammatory response in GCA. In particular, IL-32 seems to be an important mediator of artery inflammation in GCA.

Expansion of intestinal CD4+CD25(high) Treg cells in patients with ankylosing spondylitis: a putative role for interleukin-10 in preventing intestinal Th17 response.

OBJECTIVE: Subclinical gut inflammation has been demonstrated in patients with ankylosing spondylitis (AS). This study was undertaken to determine the frequency of regulatory CD4+CD25(high) T cells (Treg cells) and to evaluate Treg cell-related cytokines (interleukin-2 [IL-2], transforming growth factor ß [TGFß], and IL-10) and transcription factors (FoxP3 and STAT-5) in the ileum of patients with AS. METHODS: Quantitative gene expression analysis, by reverse transcriptase-polymerase chain reaction, of Treg-related cytokines (IL-2, TGFß, and IL-10) and transcription factors (STAT-5 and FoxP3) was performed on ileal biopsy specimens from 18 patients with AS, 15 patients with active Crohn's disease (CD), and 15 healthy subjects. Tissue and circulating Treg cells were also analyzed by flow cytometry. RESULTS: A significant up-regulation of IL-2, TGFß, FoxP3, STAT-5, and IL-10 transcripts in the terminal ileum of AS patients displaying chronic ileal inflammation was observed. Flow cytometric analysis of Treg cells showed significant peripheral expansion in both patients with AS and chronic inflammation and patients with CD (mean ± SD 1.08 ± 0.4% and 1.05 ± 0.3%, respectively) as compared with healthy subjects (0.25 ± 0.12%) (P < 0.05). Interestingly, a 5-fold increase in the proportion of Treg cells was observed in the gut of patients with AS (5 ± 3%) as compared with healthy subjects (1.2 ± 0.4%) (P < 0.001), with 70-80% of these cells also producing IL-10. In vitro studies showed that blocking IL-10 was sufficient to induce Th17 polarization on lamina propria mononuclear cells isolated from AS patients. CONCLUSION: Our findings provide the first evidence that an active Treg cell response, mainly dominated by IL-10 production, occurs in the gut of AS patients and is probably responsible for the absence of a clear Th17 polarization in the ileum of AS patients.

Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media.

Secreted proteins are of tremendous biological interest since they can act as ligands for receptors to activate downstream signalling cascades or be used as biomarkers if altered abundance is correlated with a specific pathological state. Proteins can be secreted either as soluble molecules or as part of extracellular vesicles (i.e., exosomes or microvesicles). The complete proteomic profiling of secretomes requires analysis of secreted proteins and extracellular vesicles. Hence, the method described here enriches for microvesicles, exosomes, and secreted proteins from conditioned media using differential centrifugation. The three fractions are then analyzed by mass spectrometry-based proteomics for in-depth characterization and comparison of the protein secretome of cell lines.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

The emerging role of extracellular vesicles as biomarkers for urogenital cancers.

The knowledge gained from comprehensive profiling projects that aim to define the complex genomic alterations present within cancers will undoubtedly improve our ability to detect and treat those diseases, but the influence of these resources on our understanding of basic cancer biology is still to be demonstrated. Extracellular vesicles have gained considerable attention in past years, both as mediators of intercellular signalling and as potential sources for the discovery of novel cancer biomarkers. In general, research on extracellular vesicles investigates either the basic mechanism of vesicle formation and cargo incorporation, or the isolation of vesicles from available body fluids for biomarker discovery. A deeper understanding of the cargo molecules present in extracellular vesicles obtained from patients with urogenital cancers, through high-throughput proteomics or genomics approaches, will aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.

Loss of the Timp gene family is sufficient for the acquisition of the CAF-like cell state.

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.