Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles

Anatoliy Samoylenko,Artem Zhyvolozhnyi,Martin Kögler,Olha Makieieva,Seppo J Vainio,Matthieu Roussey,Geneviève Bart,Sampson S Andoh,Jussi Hiltunen

doi:10.1038/s41598-021-99004-6

Abstract

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

Highlights

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize extracellular vesicle (EV) are still limited
To study to what extend hypoxia may impact secretion of EVs by the Renal Cell Carcinoma (RCC)-derived Renca cells, these cells were cultured in fetal bovine serum (FBS)-free medium for 24 h under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions
We found that number of EVs captured on chips functionalized with either CD9 or CD81 antibodies in supernatants form cells cultured under hypoxia was 3.1–3.6 times higher as compared to normoxia (Fig. 1)

Summary

Introduction

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell–cell communication, but the technologies to characterize EVs are still limited. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Recent advances in extracellular vesicle (EV) research have demonstrated that eukaryotic cells secrete spherical particles (0.03–1 μm diameter) that are enclosed by phospholipid b ilayer[3]. These vesicles typically contain various proteins, RNA species and metabolites. There are evidences that the EV subgroup named exosomes (30–100 nm), which derive from the endosomal pathway and are released by multivesicular bodies, is involved in intercellular signaling, and seems important for primary tumor growth and for formation of metastatic foci[4]. Even though EVs are promising diagnostic and therapeutic liquid biopsy components, their wide use for such purpose is essentially requiring better methods for EV isolation and characterization

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