Normal and Cancerous Tissues Release Extrachromosomal Circular DNA (eccDNA) into the Circulation

Abstract

Cell-free circulating linear DNA is being explored for non-invasive diagnosis and management of tumors and fetuses, the so-called liquid biopsy. Previously, we observed the presence of small extrachromosomal circular DNA (eccDNA), called microDNA, in the nuclei of mammalian tissues and cell lines. Now, we demonstrate that cell-free microDNA derived from uniquely mapping regions of the genome is detectable in plasma and serum from both mice and humans and that they are significantly longer (30–60% >250 bases) than cell-free circulating linear DNA (~150 bases). Tumor-derived human microDNA is detected in the mouse circulation in a mouse xenograft model of human ovarian cancer. Comparing the microDNA from paired tumor and normal lung tissue specimens reveals that the tumors contain longer microDNA. Consistent with human cancers releasing microDNA into the circulation, serum and plasma samples (12 lung and 11 ovarian cancer) collected prior to surgery are enriched for longer cell free microDNA compared to samples from the same patient obtained several weeks after surgical resection of the tumor. Thus, circular DNA in the circulation is a previously unexplored pool of nucleic acids that could complement microRNAs (miRs) and linear DNA for diagnosis and for intercellular communication.

Cell-Free eccDNAs: A New Type of Nucleic Acid Component for Liquid Biopsy?

“…A study has shown that tumor specimens contain longer eccDNAs than normal lung tissues. Consistently, the circulating eccDNAs were longer in lung cancer patients collected prior to surgical resection of the tumor than in the same patient after surgery (37). Another study revealed that methotrexate (MTX)-treated human lymphoblastoid cells lines (LCLs) generated higher numbers of unique microDNA entities compared to non-treated cells.…”

“…The enrichment is involved in exonuclease digestion (such as Plasmid-Safe-ATP-dependent DNase and/or exonuclease V) to remove linear DNA and multiple displacement amplification (MDA) to preferentially amplify circular DNAs (1, 3, 5, 6, 16, 27, 36–38). This method has been proved to be effective for eccDNA detection and enrichment, especially when the initial DNA amount is very limited, for example the cell-free DNA (cfDNA) in the circulating system (27, 37). Other reported methods for eccDNA enrichment include multiple rounds of extensive exonuclease V digestion and chloride ethidium bromide density gradient centrifugation.…”

“…It is believed that the larger eccDNAs are basically located inside of the nucleus while the smaller ones spread in nucleus, cytoplasm, and extracellular sites. Recent findings have shown highly abundant smaller eccDNAs in the form of cfDNAs in human circulation system in both healthy and cancerous conditions (27, 37). Consistent with the origin features of eccDNAs in tissues and cell lines, the cell-free eccDNAs also showed some distribution preference in the genome, such as UTRs, exons, DNase-hypersensitivity sites and histone markers-enriched regions (27, 37).…”

“…Recent findings have shown highly abundant smaller eccDNAs in the form of cfDNAs in human circulation system in both healthy and cancerous conditions (27, 37). Consistent with the origin features of eccDNAs in tissues and cell lines, the cell-free eccDNAs also showed some distribution preference in the genome, such as UTRs, exons, DNase-hypersensitivity sites and histone markers-enriched regions (27, 37). By comparing the sizes of microDNAs in circulation, studies have found that cell-free microDNAs have different sizes under different conditions.…”

“…By comparing the sizes of microDNAs in circulation, studies have found that cell-free microDNAs have different sizes under different conditions. Lung cancer and ovarian cancer patients have longer circulating eccDNAs in pre- than in post-tumor resection (37). ctDNA in cancer patients are frequently shorter than normal cfDNAs, suggesting that size selection can improve the detection of ctDNA (6, 54).…”