Simona Principe

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.

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.

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

ABSTRACT 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.

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.

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.

Abstract B34: Signaling in the tumor microenvironment: Proteomics analyses of stromal-tumor interaction in oral cancers

Paracrine signaling between cancer-associated fibroblasts (CAFs) and cancer cells creates a bidirectional and cooperative network that drives cancer growth and progression. During the development of oral squamous cell carcinoma (OSCC), alterations in the tumor microenvironment and secretion of soluble proteins from subpopulations of CAFs generate a niche that has functional implications on tumor progression. To investigate stromal heterogeneity in OSCC and identify fibroblast-associated proteins that actively contribute to oral cancer carcinogenesis, we employed a proteomics approach to uncover the secretome of patient-derived oral CAFs. Communication in the tumor microenvironment can be mediated by classically secreted molecules, as well as by the release of extracellular vesicles, which are involved in the transfer of oncogenic factors to recipient cells. Hence, we comprehensively characterized the protein content of fibroblasts-derived conditioned media (CM) and exosomes (Exo), to identify secreted, cytoplasmic or membrane-associated molecules that could function as regulators of OSCC progression. Furthermore, to investigate how secreted signals from the surrounding stroma interact with target surface receptors, we also generated a comprehensive proteomic database of highly purified plasma membrane proteins isolated from two established tongue cancer cell lines (SCC4 and SCC25). The aim of the current study is to investigate the molecular crosstalk between CAF-derived factors and epithelial oral cancer cells to improve our understanding of the complex tumor-stroma interactions. Matched pairs of human primary fibroblasts were isolated from resected tongue cancers (CAFs) and tumor adjacent tissue (AFs), characterized according to morphology, expression of myofibroblast markers (α-SMA, tropomyosin), and the ability to degrade collagen. CM and Exo were collected after 48 hours of serum deprivation and Exo were purified by differential ultracentrifugation. Plasma membrane molecules from SCCs were isolated using colloidal silica-beads followed by density gradient ultracentrifugation. Each sample was analyzed by nano-flow ultra-performance liquid chromatography (UHPLC) coupled to a Q-Exactive tandem mass spectrometer. Our proteomic analyses quantified a total of 6638 proteins, 2855 in the CAFs/NAFs secretome (CM and Exo) and 5754 in the SCCs lines (membrane depleted (MD) and plasma membrane (PM) fractions) using the MaxQuant pipeline. CM was highly enriched in fibroblast-secreted proteins such as MMPs, VIM, IGFBPs, SPARC and Gene Ontology (GO) terms related to cytoplasmic and extracellular components. The quality of the Exo purification was confirmed by the presence of known markers such as CD81, CD63, TSG101 and FLOT1 and GO enrichment for endosomal and cytoplasmic vesicle-related terms. We used a subtractive, quantitative proteomics approach to highlight a CAF-enriched exosomal cluster consisting of 255 proteins groups differentially expressed, compared to patient matched AFs. Comparative Reactome pathway analysis revealed that this cluster is significantly enriched in metabolic enzymes involved in glycolysis and catabolic processes, as well as membrane-bound signaling receptors and proteins involved in transport or vesicles trafficking. Our proteomic analyses provide a detailed overview of signaling factors, receptors and intracellular proteins, associated with the induction of a pro-invasive stroma. These findings highlight differential expression of key signal transduction molecules that have been previously associated with cancer, albeit their precise roles in cancer progression need to be further validated. Our CAF-enriched secretome signature, complemented with the plasma membrane proteomics of tongue cancer cells represent a comprehensive data resource to investigate molecular signaling mechanisms within the tumor microenvironment. Citation Format: Simona Principe, Vladimir Ignatchenko, Alexander Ignatchenko, Ankit Sinha, Keira Pereira, Laurie Ailles, Thomas Kislinger. Signaling in the tumor microenvironment: Proteomics analyses of stromal-tumor interaction in oral cancers. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B34. doi:10.1158/1538-7445.CHTME14-B34