Human cervical cancer cell lines with known HPV positivity for HPV type 16 – SiHa, HPV type 18 – HeLa; and HPV-negative C33a were originally procured from ATCC. The materials used in the study have been listed along with their source of procurement. DMEM (#AL111-18X500ML), MEM (#AT154), antibiotic solutions (#A018). The materials used in the study have been listed along with their source of procurement. DMEM (#AL111-18X500ML), MEM (#AT154), antibiotic solutions (#A018), bovine serum albumin fraction V (#RM10409) were procured from HiMedia Laboratories Pvt. Ltd. (Mumbai, India), Pierce™ BCA Protein Assay Kit (#23225), exosome-depleted serum One Shot™ format (#A2720803), Precision Plus Protein Dual Color Standards from Bio-Rad, USA (#161–0374), High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™; #4368814), TRIzol RNA Isolation Reagent (#AM9738) were procured from Thermo Fischer Scientific (Waltham, USA). ExoEnrich™ (#PEC-50), ExoLyseP™ (#PEL-25P) were purchased from ExoCan Healthcare Technologies Ltd. (Pune, India). ECL-substrate (#SC-2048) from Santa Cruz Biotechnology, Inc. (Dallas, USA). All the antibodies were procured from Santacruz Biotechnology, Inc. (USA) and Sigma (St. Louis, USA) (Supplementary Table 1) and oligos used in the study were procured from Eurofins scientific (Supplementary Table 2). Millipore PVDF membrane (#HVLP04700), RNAse A (#P4170), and all other reagents unless specified were procured from Sigma.
Preparation of cell culture conditioned media for exosome studies:
Briefly, cells were seeded at 25% confluency (9 × 105) in a 100 mm culture plate containing 10% exosome depleted-FBS and allowed to grow for 4 days. After 4 days, cell culture conditioned medium containing exosomes was harvested and centrifuged at 2000 rpm for 10 min to remove dead cells followed by centrifugation at 5000 rpm for 30 min to pellet down remaining cellular debris. The resulting supernatant was then subjected to 0.2 µm filtration step using 0.22 µm membrane (Millipore, MA, USA)
Isolation of exosomes from cervical cell culture conditioned medium
Exosomes were isolated using commercially available kit, ExoEnrich™ as described previously without any deviation. Exosomes were isolated using ExoEnrich™ (PEC-50; ExoCan Healthcare Technologies Ltd, Pune, India) as per manufacturer’s instructions. Briefly, 4 ml of culture conditioned medium was mixed with 100 µl of solution A followed by addition of 2 ml of solution B. The resulting suspension was mixed by gentle pipetting and centrifuged at 3000 rpm for 20 min. The exosome pellet was washed with 1× PBS twice at 3000 rpm, 3 times each. The exosomes were used in downstream analysis or were stored at -20◦C until further use.
Transmission electron microscopy (TEM) of cervical cancer exosomes
TEM analysis of exosome samples was performed according to previously published reports with minor modifications (29, 30). Freshly isolated cervical cancer exosomes were resuspended in 30 µl of 1X PBS containing 2% paraformaldehyde. Exosomes were prepared for TEM inspection by adsorbing onto Formvar carbon-coated nickel grid for a time period of 1 h. The grids were fixed by 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.6 for 10 min. After rinsing with sterile distilled water, the grids were contrasted using uranyl-oxalate solution at pH 7 for 5 min, air-dried for 5 min and examined with a JEOL 2100F transmission electron microscope (JEOL Ltd., Tokyo, Japan) operated at 100 kV.
Isolation of exosomal proteins and immunoblotting for exosome-specific markers
Total exosome proteins were isolated using ExoLyseP™ and immunoblotted as described earlier (31). Antibodies and their specific dilution in the blocking solution used in the study are described in Supplementary Table 1. Immuno-active bands were detected on an Amersham Imager 600 (GE Life Sciences ABI, Sweden) after 5 min treatment of the blot with enhanced chemiluminescence detection kit.
Cellular and exosomal RNA isolation and quantification
Exosome pellets were removed of exterior DNA by DNaseI digestion and treated with RNase to remove outer RNA as using TRIzol reagent as per manufacturer’s instructions. Trizol was added to 106 cells and to exosomes isolated from 4 ml of conditioned medium normalized per 106 cells from all the cell lines followed by homogenization and treatment of chloroform. The suspension was allowed to stand at RT for five minutes and then subjected to centrifugation at 12,000 rpm for 15 minutes as per manufacturer’s instructions. The aqueous layer was taken off and collected in a new tube. RNA was precipitated using isopropanol at -80°C for overnight and pelleted at 12,000 rpm for 20 min. Isolated RNA was dissolved in a minimum of 20 µl of nuclease free water. RNA was quantified spectrophotometrically using NanoQuant Tm (Tecan). For RNA visualization silver staining was performed on non-denaturing PAGE as described earlier (31). Briefly, RNA was isolated from exosome preparations (from 4 ml culture conditioned medium) and subjected to polyacrylamide gel electrophoresis for 3 h at 100 V in 0.5× Tris-borate buffer. The gel was fixed in 150 ml of 50% Methanol 5% Acetic acid for 20 min followed by washing with 50% Methanol and water for 10 min. The gel was sensitized using 0.02% sodium thiosulphate solution for 1 min followed by a silver reaction using 0.1% Silver Nitrate in 0.08% formalin (37%) for 20 min and developed using 2% sodium carbonate with 0.04% formalin. The RNA concentrations were determined by Qubit (Thermo Fisher Scientific, USA) and the RIN for sequencing analysis.
Transcript analysis by reverse transcriptase (RT)-PCR
A minimum of 2 µg of sample RNA was used for cDNA synthesis in a 20 µl reaction using High-Capacity cDNA Synthesis Kit as described previously(32). PCR was performed for amplification of specific genes on Veriti Thermal Cycler Pro from Applied Biosystems in a 10 µl reaction volume. Primer sequence with annealing temperature is described in Supplementary Table ST2.
Modulation of exosomal HPV RNA cargo by cytotoxic drugs: HPV-positive cervical cancer cells were exposed to cytotoxic drugs, 5-FU and cisplatin. For this, 2.5× 103 cells were seeded in a 96-well plate and grown overnight to allow attachment. Subsequently, cells were treated with IC50 doses of 5-FU (17.58 µM: SiHa; 15.31 µM: HeLa) and cisplatin (24.14 µM: SiHa; 6.82 µM: HeLa) for 48 h and the exosomes were isolated from the conditioned media of treated cells. The exosomal RNA was isolated and examined for modulation of HPV transcripts by type-specific HPVE6 and E7 RT-PCR.
RNA isolation, NGS library preparation and sequencing
RNA isolation, NGS library preparation and high-throughput sequencing was outsourced to Clevergene Biocorp. Pvt Ltd. (Bengaluru, Karnataka, India). Total RNA in the exosomes was isolated using the TRIzol reagent according to the manufacturer’s instructions. The RNA concentrations were determined by Qubit (Thermo Fisher Scientific, USA) and the RIN (RNA Integrity Number) was checked by Agilent 2100 Bioanalyzer (Agilent Technologies). For each library preparation, 1 ng of total RNA from each sample was used. NEBNext Ultra II RNA Library Prep Kit for Illumina (# E7775) was used. Total cDNA was ligated with P7 and P5 adapter sets. The PCR products were gel purified and their quality confirmed by Bioanalyzer (Supplementary Figure SF1). Paired-end sequencing was performed on these libraries using an Illumina HiSeq X platform with a minimum of 25–30 million reads per sample.
Sequence Data QC:
NGS data quality was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and MultiQC(33). The data was checked for base call quality distribution, % bases above Q20, Q30, %GC, and sequencing adapter contamination (Supplementary Table ST3). Raw sequence reads were processed to remove adapter sequences and low-quality bases using fastp(34).
Alignment and Expression Analysis
The QC passed reads were mapped onto indexed Human (GRCh38.p7), HPV16 and HPV18 reference genome using STAR v2 aligner(35). Uniquely mapped reads were used for transcripts assembly. For assembling transcripts, StringTie was used with default parameters(36). Assembled transcripts of all the samples were merged into a single gtf file using the String Tie merge option. The merged gtf was compared and annotated with reference gff using gff compare(37). The relationship between the assembled transcripts and closely related reference transcripts along with the key for these codes is provided in Supplementary Figure SF2.
Differential transcript abundance analysis
Differential analysis was carried out using the DESeq2(38). The read counts were normalized (variance stabilized normalized counts) and differential enrichment analysis was performed. For HPV-negative vs. HPV-positive analysis, C33a was used as reference and SiHa and HeLa exosomal transcripts were used as test groups. For HPV16 vs. HPV18 analysis, SiHa was used as reference and HeLa exosomal transcripts were used as test group. Transcripts with absolute log2 fold change ≥ 1.5 and p-value ≤ 0.05 were considered significant. The profile of differentially exported transcripts across the samples was evaluated using volcano plots. The transcripts that showed significant differential expression for human reference genome (GRCh38.p7) were used for Gene Ontology (GO) and KEGG pathway enrichment analysis.
GO and Pathway Analysis
Enrichment analysis for Biological Process (BP), Molecular Function (MF), Cellular Component (CC) and KEGG Pathways was performed using Cluster Profiler R Bioconductor package(39). GO and pathway terms with adjusted p-value ≤ 0.05 were considered significant. To visualize the GO enrichment results, GO plot, R package was used(40). GO plot package calculated z-score using the following formula
where up is the number of up-regulated genes in a GO term and similarly down represents number of down-regulated genes in the GO term. The z-score provides a rough idea about the expression profile of genes within a GO term. The pathways were visualized using the Pathview package to check the differential expression level of the genes in the pathway(41).