EV DNA from pancreatic cancer patient-derived cells harbors molecular, coding, non-coding signatures and mutational hotspots

Abstract

DNA packaged into cancer cell-derived EV is not well appreciated. Here, we uncovered signatures of EV DNA secreted by pancreatic cancer cells. The cancer cells and non-cancer counterparts exhibit distinct low vs. high molecular weight (LMW vs. HMW) EV DNA fragments distribution, respectively. Genome sequencing and Single Nucleotide Variants analysis revealed that 95% of reads and 94% of SNVs map to noncoding regions of the genome. Given that ~1% of the human genome represents coding regions, the 5% mapping rate to coding regions suggests a non-random enrichment of certain coding regions and mutations. The LMW DNA fragments not only set cancer cells apart, but also harbor cancer specific enrichment of unique coding regions, the top nine being FAM135B, COL22A1, TSNARE1, KCNK9, ZFAT, JRK, MROH5, GSDMD, and MIR3667HG. Additionally, the cancer cells' LMW DNA fragments exhibit dense centromeric mapping more strikingly on chromosomes 3, 7, 9, 10, 11, 13, 17, and 20. Mutational profiling turned up close to 200 mutations specific for the cancer cells. Altogether, our analyses suggest that centromeric regions might hold clues to EV DNA content from pancreatic cancer, the molecular, mutational signatures thereof, and rationalizes the need for a new approach to DNA biomarker research.

Fig. 1. Characterization of the vesicles by Nanoparticles Tracking Analysis, transmission electron microscopy, and antibody array.

A EVs from H6C7 (i), Panc1 (ii), and HPAF-II (iii) cells isolated by the indicated methods were analyzed for size distribution by NanoSight (NTA) or B visualized with Transmission electron microscopy (TEM); scale bar is 500 nm. The bottom panels represent the zoom-in on the boxed area in the image. C Exosomes antibody array was carried out on the EVs from the cells above isolated by the indicated methods, showing expression of various EV protein markers.

Fig. 2. Distinct DNA fragments packaged in EVs released from pancreatic cancer and non-cancer cells.

A Following DNA extraction from the EVs prepared by the kit-based method, a dsDNA-specific detection method was used to validate the presence of DNA molecules in the samples; representative of 3 experiments. Equal amount of dsDNA, 2 ng, was loaded on the Agilent 4200 TapeStation system using the genomic (B) or the D5000 (D) screen tape protocol; experiments were done at least three times. C The electropherograms showing distinct peaks for the control and cancer cells; 3 representatives from the 10 cell lines. E EV preparations were treated with DNAse I before DNA was extracted. A dsDNA-specific detection method was used to confirm the presence of DNA molecules as in (A). F–H The DNA fragments in samples from (E) were analyzed as described in (B, D). The activity of the DNAse I was confirmed by analyzing EV DNA from H6C7 (#1) or MiaPaCa2 (#2) digested with the DNAse I.

Fig. 3. Whole genome sequencing analysis demonstrated a large proportion of EV DNA molecules from non-coding regions of the genome.

A Schematic diagram of the size selection. Following the sequencing of the DNA from the EVs (isolated by the kit-based method), the reads were re-aligned against the human genome, the percentage of reads from the high (B) or low (C) molecular weight fraction mapping to the coding or non-coding regions was determined bioinformatically, and graphed. D Average of the reads mapping to coding vs. non-coding regions from all ten cell lines.

Fig. 4. The low molecular weight DNA fragments distinguish the cancer cells from their non-cancer cells counterparts.

A, B Pearson correlation coefficient showing that the LMW fragments (A) are more distinctive between the cancer cells and the non-cancer cells. C, D Principal components analysis showing that the LMW fragments (C) from the cancer cells cluster away from the non-cancer cells.

Fig. 5. Unique coding DNA signatures for the LMW fragments.

Volcano plot showing the differential abundance of the DNA fragments mapping to their corresponding coding genes.

Fig. 6. The low molecular weight DNA fragments demonstrate dense centromeric mapping for the cancer cells.

A A mapping of the reads showing a dense centromeric mapping of all cancer cell lines but a sparse mapping for the control non-cancer cells. B A zoom-in of the centromeric region demonstrating clear distinct mapping signatures for the cancer vs. control cells.

Fig. 7. Mutational analysis reveals cancer cell-specific mutations in low-molecular-weight DNA fragments.

Number (A) or percentage (B) of variants that fall into coding (intragenic) or non-coding (intergenic) regions of the genome. Types of mutations, single nucleotide variant and frequency (C) or insertion, deletion, and proportion (D).