Data Collection and Bioinformatics Analysis
To obtain candidate genes for CTC and TDE in colorectal cancer, two studies (GSE31023 and GSE72577) were selected (https://www.ncbi.nlm.nih.gov/geo/); the Barbazan et al study contained six cancer and three healthy samples (40) and the Dou et al study encompassed three colorectal cancer cell lines (DLD-1, DKO-1 and DKs-8) and the corresponding TDEs (41). First, we compared markers expressed in all three TDE and CTC, but not the cell lines. The genes common to TDEs and CTCs were selected.
Characteristic biological features of selected genes were assigned according to gene ontology analysis (GO) (42, 43) to molecular functions, biology processes and cellular components using EnrichR (amp.pharm.mssm.edu/Enrichr/), STRING (https://string-db.org/). DisGeNET RDF v7.0 (ref: https://academic.oup.com/nar/article/48/D1/D845/5611674) was used to search for disease-associated markers, with emphasis on tumor growth. Disease classes were ranked based on the gene-disease associated (GDA) score and tumor growth-associated markers significantly above the mean score (0.06198) were included in the network analysis, generated using Cytoscape Version 3.7.1 (44) with ClueGO plugin (http://apps.cytoscape.org/apps/cluego) (45) based on gene ontology (GO) (http://geneontology.org/) and pathways, including Kyoto Encyclopedia of Genes and Genomes (KEGG) (https://www.genome.jp/kegg/) (46), Reactome (https://reactome.org/) (47), and WikiPathways (https://www.wikipathways.org/index.php/WikiPathways) (48). DisGeNET integrates both expert-curated databases with text-mined data, covering information on Mendelian and complex diseases (49). All analyses were performed in the R programming language to reach common molecular markers between CTC and TDE.
Cell Culture and Generation of CSC-Enriched Spheroids
The colorectal adenocarcinoma HT-29 cell line was obtained from Iranian Biological Research Center (IBRC). Colonosphere formation was carried out as described (50). Briefly, HT-29 cells were grown to 70–90% confluence. After washing with pre-warmed phosphate-buffered saline (PBS), cells were detached by trypsin/ EDTA (Gibco, Germany) and single cells were seeded in poly‐HEMA (Sigma, USA)-coated plates (low attachment condition) in DMEM/F12 serum‐free medium (Gibco, Germany) supplemented with 10 ng/ml of recombinant basic fibroblast growth factor (bFGF, PeproTech, USA), human recombinant epidermal growth factor (EGF, PeproTech, USA), 2% B27 supplement (Gibco, Germany), 2mM L‐glutamine (Gibco, Germany), 1% nonessential amino acid (Gibco, Germany), and 1% penicillin–streptomycin (Gibco, Germany). The culture medium was supplemented with bFGF, EGF, and 2% B27 supplement every third day. After ten days, culture supernatant was collected for exosome isolation.
For HT-29 cells culture, exosome‐free FBS was obtained after overnight ultracentrifugation of FBS (Gibco, Germany) at 110,000 g, 4°C (45Ti rotor, Beckman Coulter, Fullerton, California). Cells were cultured in DMEM/high glucose media (Gibco, Germany) supplemented with 10% exosome‐free FBS, 1% L‐glutamine (Gibco, Germany), 100 U/mL of penicillin, and 100 mg/mL streptomycin (Gibco, Germany), and maintained at 37°C, 5% CO2 in a humidified incubator. For exosome isolation, the culture supernatant was harvested at ~90% confluence.
Clinical Sample Collection
Peripheral blood samples were collected from 20 CRC patients and 10 healthy controls at Bahman and Firozgar hospitals from 2018-2020 under ethical committee approval. Healthy controls were enrolled from people who underwent a routine health checkup without disease detection. Cell-free plasma was isolated from all blood samples using 2000×g for 10min and suspended in Qiazole (Qiagen, Germany). Samples were stored at -80°C. Patient information including gender, age, TNM stage, tumor differentiation was also recorded.
Exosome Isolation by Ultracentrifugation
Exosomes were isolated from culture media of the HT-29 cell line, HT-29-derived spheroids and plasma samples from patients and healthy controls using ultracentrifugation. In brief, culture supernatants and plasma samples were centrifuged at 350× g for 10 min and then at 3000×g for 10 min to remove cell debris. To separate MV from other extracellular vesicles, supernatants were centrifuged at 21000×g for 20 min and the pelleted microvesicles were resuspended in PBS and stored in -80°C. The supernatant was passed twice through ultracentrifugation at 110,000g for 120 min (45Ti rotor, Beckman Coulter). The exosome pellets were resuspended in 1ml PBS or lysis buffer (51).
Scanning Electron Microscopy (SEM)
Isolated exosomes were fixed in 2.5% (w/v) glutaraldehyde for 20 min, washed in PBS and were dehydrated using a gradient of ethanol (60%, 80%, 90% and 100%). The exosomes were dried at room temperature for 10 min on glass. To make surface conductive, a coating of 2-5 nm gold-palladium alloy was applied by sputtering (SPI-Module Sputtering, Argon as gas for plasma) before imaging by SEM (EM3200, KYKY and SEM, Seron Technology, AIS‐2100, Korea).
Dynamic Light Scattering (DLS) and Protein Concentration Measurement of Exosomes
To determine the size distribution of isolated exosomes, 50 μl of exosome samples were added to 950 μl PBS and analyzed by dynamic light-scattering measurements (Malvern, UK). Protein quantification was performed by a Bicinchoninic acid assay (BCA) protein assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific).
Western Blot Analysis (WB)
Lysed samples (in RIPA Lysis and Extraction buffer) were subjected to 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, transferring the separated proteins to polyvinylidene difluoride (PVDF) membranes (Bio‐Rad, Hercules, California). After blocking, blots were incubated overnight with primary antibodies (CD9 (Santacruz, Germany (Cat No. sc-13118)), CD81 (Santacruz, Germany (Cat No. SC-166029)), TSG101 (Gentex, U.S.A. (Cat No. GTX70255)) ; 1:500), followed by a two‐hour incubation with secondary antibody (goat anti‐mouse, Invitrogen, USA). Enhanced chemiluminescence substrate (ECLTM, Thermo Fisher Scientific, USA) was used as detection reagent.
RNA Isolation and qRT-PCR
RNeasy Micro Kit from Qiagene (Qiagen Cat No. /ID: 74004) was used to isolate the total RNA from exosomes. To remove genomic DNA contamination, RNA samples were treated with DNase I and then Nanodrop (ThermoFisher Scientific, USA) was used for RNA quantification and the purity was checked by the A260/A280 ratio. Twenty nanogram of total RNAs were used for reverse transcription (RT) to generate cDNAs using PrimeScript RT Reagent Kit (Takara, Japan). SYBR Green real-time master mix was used for qRT-PCR. The corresponding primers were acquired from SinaClon company (Iran) as follow: GAPDH: 5′-AACTTTGGCATTGTGGAAGG-3′ F and 5′-CACATTGGGGGTAGGAACAC-3′ R. PDGFA: 5′-GCC CAT TCG GAG GAA GAG AA-3′ F and 5′- CAG ATC AGG AAG TTG GCG GA -3′ R. RAF1: 5′- GGT GAT AGT GGA GTC CCA GC -3′ F and 5′- GGT GAA GGC GTG AGG TGT AG -3′ R. The expression levels of PDGFA and RAF1 mRNAs were normalized by GAPDH mRNA levels based on the 2−ΔΔCt approach (52).
SPSS software version 22.0 (IBM Corp, USA) was utilized to analyze the data. GraphPad Prism version 8.0 for Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com) was used to determine the differences between tumor and normal blood samples. Pearson’s χ2 and Spearman’s correlation tests were used to analyze the significance of associations and correlations between PDGFA as well as RAF1 expression and clinicopathological parameters. Kruskal-Wallis and Mann-Whitney U tests were applied for pairwise comparisons between groups. In all parts, quantified data are derived from 20 tumors, 10 healthy and cell line-derived exosomes samples, a p-value of <0.05 was considered statistically significant. As noted, in the first step, all quantified data was replicated an average of three times.
The research ethics committee of Iran University of Medical Sciences issued (IR.IUMS.REC 1395.9221513203) for this study. All procedures including informed consent before surgery from all participants were in accordance with the abovementioned ethical standards. The Ethics Committee of the Bahman and Firozgar Hospitals approved the use of clinical samples. Besides, the patients/participants provided their written informed consent to participate in this study.