Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva.

Jieping Yang,David T W Wong,Fang Wei,Christopher Schafer,Muy-Teck Teh

doi:10.1371/journal.pone.0110641

Jieping Yang, David T W Wong + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0110641

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Journal: PloS one	Publication Date: Nov 14, 2014
Citations: 160	License type: CC BY 4.0

Affiliation: University of California, Los Angeles

Abstract

The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell–specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

Highlights

Human saliva is a clear, slightly acidic biofluid known to contain a diverse range of proteins [1], over 3000 unique mRNAs [1,2,3], and 700 bacterial species [4]
Previous studies have demonstrated that cell-free saliva supernatant contains more than 3000 different mRNA species [1,2,3], and the extracellular RNAs (exRNAs) remain stable unless they are exposed to detergents [46]
Quantitative PCR analysis of extracted RNAs revealed no notable differences in Ct values of the reference genes GAPDH, ß-actin, or RPS9 between cell-free saliva, salivary exosome-like microvesicles (ELMs), and salivary ELMs with RNase treatment (Fig. 1e). 1% Triton X-100 (Tx) solution compromised structural integrity of salivary ELMs (Fig. 1d)

Summary

Introduction

Human saliva is a clear, slightly acidic (pH 6.0–7.0) biofluid known to contain a diverse range of proteins [1], over 3000 unique mRNAs [1,2,3], and 700 bacterial species [4]. Formed from the invagination of intracellular vesicles, exosomes often contain biologically active host cell lipids, proteins, miRNAs, mRNAs, ncRNAs, and other cellular constituents [14,16,17, 18,19,20,21]. Many types of cancer cells release ELMs and tumor-derived ELMs carry a wide range of nucleic acids, including miRNA, mRNA, ncRNA and DNA [21]. Previous investigations have revealed that tumors are often the primary source of circulating membrane vesicles and increased amount of tumor derived protein, RNA and DNA were found in the blood of cancer patients [30,31,32,33]

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