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Genome Sequencing Analysis of Extracellular Vesicle DNA from Pancreatic Cancer Patient-derived Cells Reveals Coding, Non-coding Signatures and Mutational Hotspots

Cancer Genetics and Therapeutics
  • Primary Categories:
    • Basic Research
  • Secondary Categories:
    • Basic Research
Introduction:
Extracellular vesicles (EV) are reservoirs of biomolecules with pleiotropic effects on cellular functions in both normal physiology and disease state such as cancer. EVs regulate various aspects of cancer and have been touted to harbor potential diagnostic markers for cancer. To reach that diagnostic milestone and achieve an effective clinical operationalization of EVs, a better characterization of EVs molecular content is imperative. In particular, it is hypothesized that DNA, as a candidate EV molecule, is a better fit for a non-invasive disease diagnosis. This is because DNA is evolutionarily a more stable biomolecule in which genetic information is stored. The stability of DNA is critical and desirable not just for ensuring the preservation and accurate transmission of genetic information but also for a potential use as disease diagnostic marker. Though, to be used as such, it is central to explore cell-specific EV DNA features that set apart different cell types, in this case cancer cells vs. non-cancer cell.

Methods:
EVs were collected from pancreatic cancer cells and non-cancer counterpart, and substantiated by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy, and antibody array. The Agilent 4200 TapeStation system was then used to analyze DNA fragments distribution, followed by genome sequencing of the EV DNA fragments, reads mapping, and Single Nucleotide Variants (SNVs) analysis.

Results:
DNA from the cancer cells and non-cancer counterparts exhibit distinct low vs. high molecular weight (LMW vs. HMW) DNA fragments distribution, respectively. Genome sequencing reveals that 95% of reads and 94% of SNVs map to noncoding regions of the genome. Given the consensus that only ~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 differentiate cancer vs. non cancer cells, 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 variants specific for the cancer cell-derived LMW DNA fragments.

Conclusion:
Our analyses point to centromeric non-coding regions as holding clues to pancreatic cancer EV DNA content, the molecular, and mutational signatures thereof. Further, the study rationalizes the need for a new approach to DNA biomarker discovery.

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