Nicole Nieman

Introduction. The gold standard for tumor diagnosis is currently an invasive and expensive tissue biopsy, but due to increasing genetic sequencing technology research has investigated the potential for liquid biopsies using cell free DNA (cfDNA) to diagnose and monitor cancer^1-4. There are three main mechanisms believed to release cfDNA apoptosis, necrosis, and active secretion^2,3. Since all cells can release cfDNA it is essential to identify the tissue of origin, one potential method is analyzing methylation markers^5,6. In addition to the potential use of cfDNA in diagnosis, evidence shows tumor cfDNA is taken up by normal cells and integrates into the nuclear genome resulting in metastases formation, a process called genomemetastasis^7-8. This process may be prevented by treatment with DNase and proteinase enzymes to degrade the fragments and prevent peripheral uptake^7-9. Methods. Mouse fibroblast NIH3T3 cells were treated with fluorescently labeled cfDNA for temporal analysis. In vivo studies were performed on BALB/c mouse models. NIH3T3 cells were treated with SW480 supernatant, colorectal cancer patient serum, and control serum from healthy individuals a repeated experiment was performed with the addition of DNase I and proteinase; the cells were subsequently examined for foci formation. C57Bl/6J mice were treated with LLC cells followed by DNase, the number of metastasis was counted. Results. cfDNA is rapidly taken up by treated cell cytoplasm and nuclei⁷. In vitro and vivo treatment of mouse cells with tumor cell supernatant is associated with focus and tumor formation⁸. Larger cfDNa fragments were had a slower nuclear uptake, but were more efficient in genome integration⁷. Additionally, the larger fragments induced a greater percentage of cells to express DDR proteins and pro-apoptotic caspase 3⁷. Mice transplanted with tumor cells produced few and smaller metastasis when treated with DNase⁹. Supernatant treated with proteinase followed by DNase I was depleted of DNA did not induce tumor or focus formation⁸. Conclusions. Research has shown that cfDNA is capable of entering cells, integrating into the genome and inducing cell damage or death. cfDNa is capable of horizontal gene transfer and inducing tumor metastasis, but treatment with DNase and proteinase can decrease the role of cfDNA in genomemetasatsis. In continuing to explore the potential use of cfDNa as a liquid biopsy for cancer diagnosis and treatment monitoring, equal attention should focus on cfDNA as a potential treatment target.

1. Cheng F, Su L, Qian C. Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer. Oncotarget. 2016; 7(30):48832-48841. doi: 10.18632/oncotarget.9453.
2. Wan JC, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, Pacey S, Baird R, Rosenfeld N. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017; Advanced online publication. doi: 10.1038/nrc.2017.7
3. Thierry AR, El Messaoudi S, Gahan PB, Anker P, Stroun M. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev. 2016; 35(3):347-76. doi: 10.1007/s10555-016-9629-x.
4. Salvi S, Gurioli G, De Giorgi U, Conteduca V, Tedaldi G, Calistri D, Casadio V. Cell-free DNA as a diagnostic marker for cancer: current insights. Onco Targets Ther. 2016; 9:6549-6559.
5. Sun K, Jiang P, Chan KCA, et al. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments. Proceedings of the National Academy of Sciences of the United States of America. 2015;112(40):E5503-E5512. doi:10.1073/pnas.1508736112.
6. Lehmann-Werman R, Neiman D, Zemmour H, et al. Identification of tissue-specific cell death using methylation patterns of circulating DNA.Proceedings of the National Academy of Sciences of the United States of America. 2016;113(13):E1826-E1834. doi:10.1073/pnas.1519286113.
7. Mittra I, Khare NK, Raghuram GV, et al. Circulating nucleic acids damage DNA of healthy cells by integrating into their genome. J biosc. 2015. 40(1):91-111
8. Trejo-Becerril C, Pérez-Cárdenas E, Taja-Chayeb L, et al. Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Lichty B, ed. PLoS ONE. 2012;7(12):e52754. doi:10.1371/journal.pone.0052754.
9. Alekseeva LA, Mironova NL, Brenner EV, Kurilshikov AM, Patutina OA, Zenkova MA. Alteration of the exDNA profile in blood serum of LLC-bearing mice under the decrease of tumour invasion potential by bovine pancreatic DNase I treatment. Ahmad A, ed.PLoS ONE. 2017;12(2):e0171988. doi:10.1371/journal.pone.0171988.