DOI: 10.5061/dryad.jdfn2z393
title: Towards establishing extracellular vesicle-associated RNAs as biomarkers for HER2+ breast cancer

description: 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.
description: <strong>Bioreactor culture</strong>
 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 &lt;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. <strong>EV
 isolation and purification</strong> 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. <strong>EV visualisation by
 transmission electron microscopy (TEM)</strong>
 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). <strong>Protein analysis by western
 blotting</strong> 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). <strong>RNA quantitation by
 qRT-PCR</strong> 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.  
 <strong>Bioinformatic
 meta-analyses</strong> 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).
description: <b>Figure 1B_TEM </b> New Figure
 1B_TEM Figure 1B_image_57.tif
 --<b>Title</b>: TEM image --
 <b>Description</b>: Raw data for TEM image 
 <b>Figure 1C and 1D </b> Figure
 1C_NTA Capture MEV ExperimentReport
 --<b>Title</b>: NTA Capture MEV Experiment
 Report Figure 1D_NTA Capture SEV
 ExperimentReport --<b>Title</b>: NTA
 Capture SEV ExperimentReport --
 <b>Description</b>: 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. <b>Figure 1E qEV BCA and particle
 raw data </b> Figure 1E_qEV BCA and particle raw
 data.xlsx -- <b>Title</b>: 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. --
 <b>Description</b>: 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 <b>Figure 1F_western blot
 raw_not_cropped.pptx</b> --
 <b>Title</b>: Western blot raw images   --
 <b>Description</b>: 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). <b>Figure 2A and
 Figure S1</b> <b>Figure
 2A_RT-qPCR_rawdata.xlsx</b> --
 <b>Title:</b> Raw data for RT-qPCR to examine the mRNA
 expression level of five protein-coding genes (<i>EpCAM, BIRC5,
 YBX1, GAPDH, HPRT1</i>) and three long non-coding RNAs
 (<i>ZFAS1, HOTAIR, AGAP2-AS1</i>) in BT-474 cells and their
 EVs. -- <b>Description:</b>
 <b>For Figure 2A </b>
 <b>Sheet 1</b>: 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 <b>For Figure S1</b>
 <b>Sheet 2:</b> 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 <b>Figure 2B and
 C_meta_analysis_rawdata.xlsx</b> --
 <b>Title</b>: 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 -- <b>Description</b>: 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 -- <b>Title</b>: 201209_EVpaperScript.R -- <b>Description</b>: The R script containing the code for all the above computations and visualisations for Figure 2B and C.