In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome

doi:10.1016/j.bbrc.2013.12.070

Biochemical and Biophysical Research Communications

Volume 445, Issue 4, 21 March 2014, Pages 694-701

https://doi.org/10.1016/j.bbrc.2013.12.070 Get rights and content

Highlights

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Improved exosome proteome detection and quantification.
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2000 proteins not previously described in the Exocarta database.
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Exosome cargo enriched in functional annotations related to signalling biology.

Abstract

Molecular communication between cancer cells and its stromal microenvironment is a key factor for cancer progression. Alongside classic secretory pathways, it has recently been proposed that small membranous vesicles are alternative mediators of intercellular communication. Exosomes carry an effector-rich proteome with the ability to modulate various functional properties of the recipient cell. In this study, exosomes isolated from four epithelial ovarian cancer cell lines (OVCAR3, OVCAR433, OVCAR5 and SKOV3) were characterized using mass spectrometry-based proteomics. Using an optimized workflow consisting of efficient exosome solubilization and the latest generation of proteomic instrumentation, we demonstrate improved detection depth. Systematic comparison of our cancer cell line exosome proteome against public data (Exocarta) and the recently published NCI 60 proteome revealed enrichment of functional categories related to signaling biology and biomarker discovery.

Introduction

Epithelial neoplastic cells transition into a mesenchymal phenotype with increased motility and invasion, two fundamental hallmarks of cancer [1], [2]. These changes in phenotype allow epithelial ovarian carcinoma (EOC) cells to adhere to mesothelial cells as part of the invasion process into the peritoneal cavity. Malignant invasion of the peritoneal cavity often results in the accumulation of ascitic fluid, which can be due to obstruction of lymphatic drainage or through non-obstructive mechanisms in which EOC cells secrete biomolecular factors to propagate a cancer supporting niche [1]. This is evident through analysis of ovarian cancer ascites, containing a multitude of cancer specific/promoting factors [3]. Hence, the interplay between neoplastic cells and its stroma is one of the key factors in cancer growth and progression [4], [5]. Previous studies have shown that tumour cells can modulate their microenvironment by secreting biomolecular factors to promote angiogenesis, invasion and immunosuppression [6]. Recent studies have shown that such communication is not limited to classically secreted factors, but can also be mediated by various small extracellular vesicles, such as exosomes, which will be the primary focus of the current study. [7], [8]. Exosomes are small (40–100 nm diameter) membranous vesicles secreted through the endocytic pathway by most cell types and tumour cells. The protein content and the biological role of exosomes are dependent on the secreting cell type. For instance, exosomes derived from dendritic cells support the survival of naïve T-cells [9], whereas exosomes derived from cancer cells promote angiogenesis, stromal remodeling and transfer of growth factors and receptors to modulate signaling pathways of fibroblasts and other stromal components [10], [11]. This suggests that exosomes originating from cancer cells play a role in cancer progression and modulation of the tumor microenvironment and in-depth characterization of the protein cargo of exosomes will help to elucidate their precise biological function. Hence, we hypothesize that systematic analyses of the EOC exosome proteome and integration with available data resources will reveal enriched proteins and pathways, potentially involved in intercellular communication.

Section snippets

Cell culture

The four EOC cell lines, OVCAR3, OVCAR433, OVCAR5 and SKOV3 cell lines were grown in RPMI-1640 media supplemented with 10% fetal bovine serum, and 100 U/ml penicillin–streptomycin (all from Gibco, Canada). Cells were cultured in ten 150 mm dishes at 37 °C in a 5% CO₂ humidified incubator. Once 80% confluency was achieved, the supernatant was removed and the monolayer was rinsed twice with warm PBS followed by addition of 20 mL of serum free media (RPMI-1640 media without phenol red and 100 U/ml

In-depth proteomics of EOC-derived exosomes

The workflow for isolation and proteomics analysis of exosomes is described in Fig. 1A. Here, we starved cells for 48 h in serum-free media, which was subsequently used as the source for exosome isolation. Through differential centrifugation, cellular debris and larger microvesicles were pelleted and discarded, and high-speed ultracentrifugation (120,000×g) was used for exosome isolation. Exosome pellets were extracted using an optimized 2,2,2-trifloroethanol solubilization/digestion protocol

Discussion

The objective of this study was to present an in-depth proteomic characterization of EOC exosomes using the latest generation of chromatographic and mass spectrometric instrumentation. Exosome isolation combined with high resolution chromatography and mass spectrometry will allow for more detailed interrogation of the exosome proteome potentially leading to a better understanding of their biological function. A crucial step of the current study was to utilize a TFE-based extraction of proteins

Acknowledgments

This work was supported in part by grants from the Canadian Institute of Health Research (MOP-93772) to T.K. This research was funded in part by the Ontario Ministry of Health and Long Term Care. The views expressed do not necessarily reflect those of the OMOHLTC. T.K. is supported through the Canadian Research Chairs Program. A.S. is supported through a Medical Biophysics Excellence Award and the Kristi Piia Callum Memorial Fellowship. We would like to thank Dr. Rottapel (Princess Margaret

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View full text

In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome

Highlights

Abstract

Introduction

Section snippets

Cell culture

In-depth proteomics of EOC-derived exosomes

Discussion

Acknowledgments

Cell

Cell

Gynecol. Oncol.

Cell. Immunol.

J. Proteomics

Kidney Int.

Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research

Nat. Rev. Cancer

In-depth proteomics of ovarian cancer ascites: combining shotgun proteomics and selected reaction monitoring mass spectrometry

J. Proteome Res.

Tumor stroma and regulation of cancer development

Annu. Rev. Pathol.

Stromal cells in tumor microenvironment and breast cancer