Colorectal Cancer Cell Line SW480 and SW620 Released Extravascular Vesicles: Focus on Hypoxia-induced Surface Proteome Changes

Materials and Methods

SW480 and SW620 cell culture. The human colorectal adenocarcinoma cell lines SW480 and SW620 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in DMEM/F12 medium (Lonza, Basel, Switzerland #BE12-79F) supplemented with 10% foetal bovine serum (Sigma- Aldrich, St. Louis, MO, USA, #F7524, 1% L-glutamine (Lonza, #17-605E) and 1% antibiotic-antimycotic (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, #15240-062) in a humidified 5% CO₂ atmosphere at 37°C. The cell cultures were monitored by mycoplasma infection by using PCR Mycoplasma Test Kit I/C (PromoKine, PromoCell GmbH, Heidelberg, Germany, # PK-A91-1096). For the isolation of EV, 1×10⁶ cells per ml were seeded in serum -free DMEM/F12 medium supplemented with 1% L-glutamine (Lonza, #BE12-605E), 10 ng/ml beta fibroblast growth factor (bFGF) (Santa Cruz, Biotechnology, Dallas, TX, USA, #sc-4573), 20 ng/ml epidermal growth factor (EGF) (R&D Systems, Minneapolis, MN, USA, #236-EG-200), 50 ng/ml hydrocortisone (Sigma-Aldrich, H0135), 1×B27 (Invitrogen, Fisher Scientific, Leicestershire, UK #17504001) and grown for 48 h in suspension at hypoxic (1% oxygen, 94% nitrogen, 5% CO₂) or normoxic (20% oxygen, 75% nitrogen, 5% CO₂) conditions.

EV isolation. Culture media were collected and centrifuged at 300×g for 5 min to remove the cells and at 3,000 × g for 30 min at +4°C to remove cell debris, and then filtered through 0.2 μm filters (Sarstedt, Numbrecht, Germany, #83.1826.001). After filtration, media were concentrated up to 1 ml with 100 kDa centrifuge filters (Merck Millipore, MA, USA #UFC910024). Size exclusion chromatography (SEC) was performed to separate EV from protein aggregates by using CL6B sepharose (GE healthcare, Buckinghamshire, UK #17-0160- 01) filled columns (Kinesis, 900-0000-016T). Further, each SEC fraction was measured by Zetasizer Nano ZS and all fractions containing particles ranging in size from 30 to 200 nm were pooled and concentrated up to 100 μl by 3-kDa filters (Merck Millipore, UFC500324) and aliquoted to avoid repeated freeze/thaw cycles. Isolated EV where characterized by western blot (WB) and Transmission Electron Microscopy (TEM).

Immunofluorescence. Multicellular SW480 and SW620 spheroids were centrifuged at 300 × g for 5 min onto slides coated with poly-L-lysine, fixed with cold methanol/acetone for 20 min at −20°C, blocked with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min at room temperature. Then the slides were incubated with primary antibodies – rabbit anti- CAIX (Abcam, Cambridge, UK, #ab15086) diluted (1:50) and mouse monoclonal anti-HIF1α (R&D Systems, MAB1935) diluted (1:50) overnight at +4°C. After incubation with the primary antibodies, slides were washed and incubated for 1 h at room temperature with secondary antibodies – goat anti-rabbit IgG conjugated with fluorescein (FITC) (Jackson ImmunoResearch, Cambridgeshire, UK, #11-095-006) or goat anti-mouse IgG conjugated with cyanine (Cy3) (Jackson ImmunoResearch, 115-165-0) diluted (1:200) and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, #P36935).

Western Blot (WB). Proteins were isolated from EV with RIPA buffer. One microgram of EV total protein was loaded per lane and separated by 10% SDS-PAGE. Proteins were electro blotted onto nitrocellulose membranes and blocked with 10% (w/v) fat-free milk and then incubated with the primary antibodies: CAIX (Abcam, ab15086) (1:1,000), ALIX (Santa Cruz, sc-166952) (1:250), TSG101 (Abcam, ab125011) (1:1,000), Calnexin (Abcam, ab22595) (1:1000) and β-actin (Abcam, ab8224) (1:2000). After washing, the membranes were incubated with peroxidase - conjugated rabbit anti-mouse secondary antibody (Santa Cruz, sc-516102) (1:2000) or mouse anti-rabbit secondary antibody (Santa Cruz, sc-2357) (1:2000). Next, the membranes were washed and processed with ECL select western blotting detection reagents (GE Healthcare, RP2235) according to the manufacturer's instructions.

Proteomics of EVs. The concentration of EV proteins was measured using a Nano Drop and Pierce BCA Protein assay kit (ThermoFisher Scientific, Wilmington, DE, USA) and normalized as starting input material to the protein concentration 1 mg/ml. EV surface protein fraction was separated using XPEP Exosome Mass Spec kit following manufacturer's instructions (System Biosciences, Palo Alto, CA, USA, # XPEP100A1). The XPEP1000A-1 reagent kit comes complete to create EV surface protein “shaving” peptide libraries or complete EV peptide libraries. We have used the protocol for XPEP-Shave surface protein libraries. At the end of the protocol, EV peptide libraries eluted from 10 kD cut-off spin columns for Mass Spec sample preparation in the collection tube were 50 μl in volume with approximately 0.4 μg protein concentration (XPEP-Shave). According to the manufacturer's instructions, peptide libraries were ready to load on mass spectrometer directly. Chromatographic analyses were performed on a modular UHPLC system (Agilent 1290 Infinity series). Liquid chromatography separations were achieved by using an Extend-C18 RRHD column (2.1×50 mm, 1.8μm). Elution solvents consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in water in gradient mode at a flow rate of 0.3 ml/min. The high-resolution mass spectra were taken on an Agilent 6230 TOF LC/MS with electrospray ionization in positive mode. Mass spectra were collected over an m/z range of 300-2,500 Da. Protein structures corresponding to mass spectra were searched in UNIPROT-SPROT + UNIPROT-TREMBL and ExoCarta data basis with taxonomy - Homo sapiens (human), enzymatic cleavage=trypsin, fixed modifications=carbamidomethyl (C), variable modifications=oxidation (M), monoisotopic mass, peptide tolerance=1.5 Da, MS tolerance=0.8 Da, one missed cleavage, charge positions=1+, and 2+, instrument=ESI-QTOF. Relative protein intensity was calculated in intensity based absolute quantification scores - iBAQ (log 10) and was used for label-free quantification of particular protein (14). The obtained data from the UHPLC-TOF-MS runs were processed using Agilent MassHunter BioConfirm Software. Obtained sequences were used to search for full length proteins using standard human proteome database UniProt and MaxQuant software. To confirm that the found proteins are Ex proteins, we included a search in ExoCarta data basis. Protein subcellular localization data were obtained from the UniProt database.

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Figure 1.

Isolation and characterization of SW480 and SW620 EVs. A) Immunofluorescence analysis of SW480 and SW620 multicellular spheroids showing the induction of CAIX and HIF1α in hypoxic conditions. Red - HIF1α, green - CAIX, blue - nucleus. B) Transmission electron microscopy of EVs showing a cup shaped morphology typical to exosomes. White scale bar represents 100 nm. C) Western blot analysis of EVs markers (CAIX, ALIX, TSG101) and negative control (Calnexin) in EVs. H: Hypoxia; N: normoxia.