Extracellular vesicles (EVs) are emerging as key players in breast cancer progression and hold immense promise as cancer biomarkers. However, difficulties in obtaining sufficient quantities of EVs for the identification of potential biomarkers hampers progress in this area. To circumvent this obstacle, we cultured BT-474 breast cancer cells in a two-chambered bioreactor with CDM-HD serum replacement to significantly improve the yield of cancer cell-associated EVs and eliminate bovine EV contamination. Cancer-relevant mRNAs BIRC5 (Survivin) and YBX1, as well as long-noncoding RNAs HOTAIR, ZFAS1, and AGAP2-AS1 were detected in BT-474 EVs by quantitative RT-PCR. Bioinformatics meta-analyses showed that BIRC5 and HOTAIR RNAs were substantially upregulated in breast tumours compared to non-tumour breast tissue, warranting further studies to explore their usefulness as biomarkers in patient EV samples. We envision this effective procedure for obtaining large amounts of cancer-specific EVs will accelerate discovery of EV-associated RNA biomarkers for cancers including HER2+ breast cancer.

Bioreactor culture

To prevent bovine EVs present in foetal calf serum (FCS) from contaminating the cancer-specific EVs, we cultured BT-474 cells from ATCC (ATCC® HTB-20™ ) (seeded at 4.5 x 108 cells/mL) in 15 mL Advanced DMEM/F-12 medium (Gibco, ThermoFisher Scientific, Waltham, USA) supplemented with 2% CDM-HD serum replacement (FiberCell Systems, New Market, USA) in the lower cell chamber of a CELLine AD 1000 bioreactor flask (Argos, Elgin, USA). The same media (150 mL) was used in the upper media chamber but supplemented with 2% FCS (Figure 1A). The dialysis membrane that separates the cell and media compartments allows FCS-specific nutrients <10 kDa but not EVs to pass through and nourish the cells. Every three to four days, the 15 mL of conditioned medium from the cell chamber was harvested for EV isolation, and the media from the upper chamber was replaced.

EV isolation and purification

EVs were isolated using differential centrifugation and size exclusion chromatography (SEC) as outlined in Figure 1. Conditioned medium (15 mL) was first centrifuged at 2,000 x g for 10 min to remove large debris, 10,000 x g for 30 min to isolate large EVs, and 100,000 x g for 70 min to isolate small EVs (Figure 1A). The resulting small EV suspension (in 500 µL PBS) was loaded onto a 35 nm qEV original size exclusion column (Izon, Christchurch, New Zealand), and fractions 7 through 24 were collected using an automated fraction collector (500 µL per fraction). BCA protein quantitation assay (Cat # 23225, Pierce, ThermoFisher Scientific, Waltham, USA) and Nanosight NS300 nanoparticle tracking analysis (NTA; Malvern Panalytical, Malvern, UK) were performed to quantitate protein and particle concentrations in each fraction, respectively. EV concentrations and size distributions were quantified on NTA by recording three 30 seconds videos under low flow conditions, with large EVs diluted at 1:100 in PBS and small EVs diluted at 1:500 in PBS. Small EV-rich fractions (7-11) were pooled, quantified again using NTA and BCA, and concentrated by ultracentrifugation (Avanti, Beckman Coulter, Brea, USA) at 100,000 x g for 70 min.

EV visualisation by transmission electron microscopy (TEM)

Negative staining TEM of pooled EV fractions was conducted by adsorption onto Formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, USA) for 2 min, then treated with 2% uranyl acetate for 2 min. Grids were then visualised on a Tecnai G2 Spirit TWIN (FEI, Hillsboro, OR, USA) transmission electron microscope at 120 kV accelerating voltage and images were captured using a Morada digital camera (SIS GmbH, Munster, Germany).

Protein analysis by western blotting

This procedure was carried out as described previously.26 Breast cancer cell lines were grown to log-phase, washed twice with ice-cold PBS, and lysed in an sodium dodecyl sulphate (SDS) lysis buffer [60 mM Tris-HCl (pH 6.8 at 25°C), 2% (w/v) SDS, 10% glycerol]. Proteins (25 μg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Membranes were subsequently immunoblotted with antibodies recognising human HER2 (mouse monoclonal, anti-Neu, Santa Cruz, Cat # sc-33684, RRID:AB_627996), human EpCAM (rabbit monoclonal, AbCAM, Cat # ab223582, RRID:AB_2762366), human alpha-Tubulin (mouse monoclonal, Sigma-Aldrich Cat# T6074, RRID:AB_477582) and human TSG101 (rabbit polyclonal, AbCAM, Cat # ab30871, RRID:AB_2208084) and corresponding secondary antibodies. Bound antibodies were visualized using Pierce™ ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, USA) and the chemiluminescence was measured using a BioRad ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc., Hercules, USA).

RNA quantitation by qRT-PCR

Trizol-purified RNA were reverse transcribed into cDNA using qScript Flex cDNA kit (Cat # 95049, Quantabio, Beverly, USA) primed with equal molar ratio of oligo-dT and random primers according to the manufacturer’s instructions. Quantitative RT-PCR was carried out using SYBR Green MasterMix (Life Technologies, Carlsbad, USA) and gene-specific primers previously validated in the literature (Table 1). These included protein-coding mRNAs EpCAM,21 BIRC5,22 YBX1,23 GAPDH, and HPRT1, and lncRNAs ZFAS1,17 HOTAIR,19 and AGAP2-AS1.20 Three independent experiments were performed with duplicate PCR reactions per sample.  RT-qPCR data were presented as cycle threshold (CT) values. Expression values were normalized relative to GAPDH mRNA expression. Statistical analysis was performed using multiple T-test.  

Bioinformatic meta-analyses

For this meta-analysis, the “RSEM expected count (DESeq2 standardized)” dataset was downloaded on 31st March 2020 from the TCGA_GTEx_TARGET cohort included in the UCSC Xena portal (https://xenabrowser.net/datapages/) and was manually annotated. This procedure has resulted in a dataset called “Figure 2B and C_meta_analysis_rawdata.xlsx” deposited in the DRYAD Digital Repository and used for all subsequent analyses. All data manipulations, plotting and statistical analyses were carried out in R computing environment (v 3.5.3) running in R Studio (v 1.1.414) on a Windows 10 x64 machine. The ggplot2 package (v 3.3.0) was used to render Figures 2B and 2C. Magnitude of the gene expression difference between non-tumour breast tissues and breast tumours (Hedges g effect size) was calculated using the cohen.d function included in the effsize R package (v 0.8.0). The R script containing the code for all the above computations and visualisations is available in the DRYAD Digital Repository.

An earlier version of this article can be found on bioRxiv (doi: https://doi.org/10.1101/2020.09.27.309252).

Figure 1B_TEM

New Figure 1B_TEM

Figure 1B_image_57.tif

--Title: TEM image

-- Description: Raw data for TEM image 

Figure 1C and 1D

Figure 1C_NTA Capture MEV ExperimentReport

--Title: NTA Capture MEV Experiment Report

Figure 1D_NTA Capture SEV ExperimentReport

--Title: NTA Capture SEV ExperimentReport

-- Description: Raw data from hydrodynamic diameter distribution profiles of isolated large and small EVs measured by nanoparticle tracking analysis (NTA) with red vertical lines and blue numbers denote standard deviation and diameters at specific peaks, respectively.

Figure 1E qEV BCA and particle raw data

Figure 1E_qEV BCA and particle raw data.xlsx

-- Title: EV concentration determined by NTA, and protein levels determined by BCA assay of fractions acquired during separation on a qEV Original size exclusion chromatography (SEC) column.

-- Description:

Each row of the spreadsheet represents one sample. Column data are described below:

column A: qEV column fraction

column B: protein concentration (ug/20ul) measured by BCA

column C: protein concentration converted to (ng/20ul)

column D: protein concentration converted to (ng/ul)

column E: EV concentration (particle/ml) measured by Nanosight

column F: Total number of particles

column G: Particles mean measured by Nanosight

column H: Particles mode measured by Nanosight

Figure 1F_western blot raw_not_cropped.pptx

-- Title: Western blot raw images  

-- Description: antibodies recognising human HER2 (mouse monoclonal, anti-Neu, Santa Cruz, Cat # sc-33684, RRID:AB_627996), human EpCAM (rabbit monoclonal, AbCAM, Cat # ab223582, RRID:AB_2762366), Tubulin (    and human TSG101 (rabbit polyclonal, AbCAM, Cat # ab30871, RRID:AB_2208084). (Sigma).

Figure 2A and Figure S1

Figure 2A_RT-qPCR_rawdata.xlsx

-- Title: Raw data for RT-qPCR to examine the mRNA expression level of five protein-coding genes (EpCAM, BIRC5, YBX1, GAPDH, HPRT1) and three long non-coding RNAs (ZFAS1, HOTAIR, AGAP2-AS1) in BT-474 cells and their EVs.

-- Description:

For Figure 2A

Sheet 1: Each row of the spreadsheet represents Ct value from one sample (duplicate samples). Column data are described below:

column A: sample =  gene name

column B,C,D: Ct values for qRT-PCR from BT474 cells

column E,F,G: Ct values for qRT-PCR from BT474 EVs

For Figure S1

Sheet 2: Each row of the spreadsheet represents expression normalised to GAPDH for Figure S1.

Row 3: sample = gene name.

Column data are described below:

Row 5,6,7: Relative gene expression normalised to GAPDH

Figure 2B and C_meta_analysis_rawdata.xlsx

-- Title: DeSeq2 normalised log2 (x+1) expression values of 10 genes in 8,867 tumours and 6,874 normal tissues downloaded on 31st March 2020 from the UCSC Xena portal using the address https://toil.xenahubs.net/download/TCGA-GTEx-TARGET-gene-exp-counts.deseq2-normalized.log2.gz

-- Description: Each row of the spreadsheet represents one sample. The first 4 columns contain sample annotations and the subsequent 10 columns hold the actual gene expression values. Column data are described below:

column A: sample =  sample ID

column B: cat2 = organ/tissue classification

column C: X_study = non-cancer (GTEX) or cancer (TCGA) classification

column D: catN2a = specific organ or cancer type categorisation including the total number of samples within each category

columns E – N: expression values of genes that are designated as official HGNC symbols

-- Title: 201209_EVpaperScript.R

-- Description: The R script containing the code for all the above computations and visualisations for Figure 2B and C.