Metabolomic Analysis of Small Extracellular Vesicles Derived from Pancreatic Cancer Cells Cultured under Normoxia and Hypoxia

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.


Metabolomic Analysis of Small Extracellular Vesicles Derived from Pancreatic Cancer Cells Cultured under Normoxia and Hypoxia
This PDF file includes: S1 Materials and Methods Figure S1, S2 Table S1-3 S1. Materials and Methods

Isolation of Cellular RNA and mRNA Analyses
PANC-1 cells (ATCC, Manassas, VA, USA) were seeded in 6-well plates at 2.0 × 10 5 cells/well and precultured in RPMI 1640 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% (v/v) fetal bovine serum (FBS, Biowest, Nuaillé, France), antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B, Nacalai tesque, Kyoto, Japan) under normoxic conditions for 24 h. The cells were washed twice with Dulbecco's phosphate-buffered saline (D-PBS, Nacalai Tesque). Thereafter, the medium was changed to advanced RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 2 mmol/L glutamine (Thermo Fisher Scientific) and antibiotics. The cells were then cultured for 48 h under hypoxic (1% O2) or normal oxygen conditions. After washing with D-PBS, the cells were sampled and stored at −80 °C. mRNA was extracted with TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer's protocol. For cDNA synthesis, a cDNA synthesis kit (ReverTra Ace α, Toyobo, Osaka, Japan) was used. Quantitative real-time PCR was performed using TB Green Premix Ex Taq II (Takara Bio, Shiga, Japan) on a StepOnePlus Real-time PCR system (Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitation was performed using the ∆∆Ct method, with the expression of RPL27 used as an internal reference. The primers used for real-time PCR are shown in Table S3.
We measured the number of particles and size distribution by nanoparticle tracking analysis (NanoSight LM10, Malvern Analytical, Malvern, UK) (biological n = 3, technical n = 3).

miRNA Analysis of sEVs
Isolation of exosomal miRNAs was performed using the miRNeasy Mini Kit (Qiagen, Venlo, Netherlands). The sEV pellets were dissolved in 100 μL of physiological saline. Five hundred microliters of QIAzol lysis reagent (Qiagen) was added to the sample. After 5-min of incubation, 5 μL of 100 pmol/L syn-cel-miR-39 (Qiagen) was added to the tube as a spike-in control for losses in preparation. The subsequent steps were performed according to the manufacturer's instructions. For cDNA synthesis, the Taqman MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) was used. Quantitative real-time PCR was performed using the TaqMan MicroRNA Assay (Thermo Fisher Scientific) and Taq-Man Universal PCR Master Mix, no AmpErase UNG (Thermo Fisher Scientific) on a StepOnePlus Real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. Quantitation was performed using the ∆∆Ct method, with synthetic spike control (syn-cel-miR-39) used as an invariant control. miRNA data were normalized by the number of particles determined by NanoSight analysis.

Transmission Electron Microscopy
Transmission electron microscopy (TEM) analysis was performed by Tokai Electron Microscopy, Inc. The sEV samples were absorbed onto carbon-coated copper grids (400 mesh) and were stained with 2% phosphotungstic acid solution (pH 7.0) for 15 s. The grids were observed using a transmission electron microscope (JEM-1400 plus, JEOL Ltd., Tokyo, Japan) at an acceleration voltage of 100 kV. Digital images were taken with a CCD camera (EM-14830RUBY2, JEOL Ltd.). Figure S1. Validation of small extracellular vesicles (sEVs) derived from PANC-1 cells cultured under normoxia and hypoxia. Immunoblotting of CD63, CD81, syntenin-1, and calnexin in PANC-1 cells and sEVs under normoxia and hypoxia. sEV markers, CD63, CD81, and syntenin-1; EV negative protein marker, calnexin. The protein content was 10 μg for the whole cell lysate (WCL) sample and 1.5 μg for the sEV sample.