Detection of circulating tumor DNA of B16-F10 melanoma syngenic model in C57BL6 mice plasma

Background. Plasma circulating tumor DNA (ctDNA) is a potential marker for tumor process monitoring. However, the feasibility of using mouse syngeneic subcutaneous melanoma model to assess ctDNA levels remains unclear.

Aim. To evaluate the feasibility of ctDNA detection in mouse B16-F10 melanoma model using droplet digital polymerase chain reaction (ddPCR).

Materials and methods. We developed and validated a ddPCR assay to quantify plasma ctDNA from B16-F10 cells. To form experimental tumors, C57Bl6 mice were inoculated with B16-F10 cells. On the first, 7th, 14th , 21st days after tumor inoculation blood was collected by retroorbital sinus puncture. ctDNA was extracted from blood plasma. On the 21st day after tumor inoculation mice were sacrificed.

Results. We validated a ddPCR assay to quantify plasma ctDNA in B16-F10 melanoma syngeneic model in C57Bl6 mice. The assay linear range was 0.5–32 copies/ul, R2 = 0.997. The empirical limit of detection of ctDNA was 1 copy/ul in 5 ng normal tissue genomic DNA background. The coefficient of variation values ranged from 44.5 % (1 copy/ul) to 16.6 % (16 copies/ul). Plasma ctDNA was detected on 21st day after tumor inoculation in B16-F10 melanoma syngeneic subcutaneous model (p = 0.004). ctDNA concentration positively correlated with tumor volume (ρ = 0.95, p = 0.05) and total circulating DNA concentration (ρ = 1, p = 0.0).

Conclusion. B16-F10 melanoma syngeneic subcutaneous model in C57Bl6 mice can be used to monitor cDNA in studies of new approaches for melanoma treatment.

1. Tivey A., Britton F., Scott J.-A. et al. Circulating tumour DNA in melanoma – clinic ready? Curr Oncol Rep 2022;24(3):363–73. DOI: 10.1007/s11912-021-01151-6

2. Bustamante P., Tsering T., Coblentz J. et al. Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma. J Exp Clin Cancer Res 2021;40(1):196. DOI: 10.1186/s13046-021-01984-w

3. Muhanna N., Eu D., Chan H.H.L. et al. Cell-free DNA and circulating tumor cell kinetics in a pre-clinical head and neck cancer model undergoing radiation therapy. BMC Cancer 2021;21(1):1075. DOI: 10.1186/s12885-021-08791-8

4. Ruhen O., Lak N.S.M., Stutterheim J. et al. Molecular characterization of circulating tumor DNA in pediatric rhabdomyosarcoma: A feasibility study. JCO Precis Oncol 2022;6: e2100534. DOI: 10.1200/PO.21.00534

5. Eun Y.-G., Yoon Y.J., Won K.Y., Lee Y.C. Circulating tumor DNA in saliva in an orthotopic head and neck cancer mouse model. Anticancer Res 2020;40(1):191–9. DOI: 10.21873/anticanres.13940

6. Rakhit C.P., Trigg R.M., Le Quesne J. et al. Early detection of pre-malignant lesions in a KRASG12D-driven mouse lung cancer model by monitoring circulating free DNA. Dis Model Mech 2019;12(2):dmm036863. DOI: 10.1242/dmm.036863

7. Thierry A.R., Mouliere F., Gongora C. et al. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res 2010;38(18):6159–75. DOI: 10.1093/nar/gkq421

8. Terasawa H., Kinugasa H., Nouso K. et al. Circulating tumor DNA dynamics analysis in a xenograft mouse model with esophageal squamous cell carcinoma. World J Gastroenterol 2021;27(41):7134–43. DOI: 10.3748/wjg.v27.i41.7134

9. Gasparello J., Allegretti M., Tremante E. et al. Liquid biopsy in mice bearing colorectal carcinoma xenografts: Gateways regulating the levels of circulating tumor DNA (ctDNA) and miRNA (ctmiRNA). J Exp Clin Cancer Res 2018;37(1):124. DOI: 10.1186/s13046-018-0788-1

10. Wei L., Xie L., Wang X. et al. Circulating tumor DNA measurement provides reliable mutation detection in mice with human lung cancer xenografts. Lab Invest 2018;98(7):935–46. DOI: 10.1038/s41374-018-0041-8

11. Labgaa I., von Felden J., Craig A.J. et al. Experimental models of liquid biopsy in hepatocellular carcinoma reveal clonedependent release of circulating tumor DNA. Hepatol Commun 2021;5(6):1095–105. DOI: 10.1002/hep4.1692

12. Olson B., Li Y., Lin Y. et al. Mouse models for cancer immunotherapy research. Cancer Discov 2018;8(11):1358–65. DOI: 10.1158/2159-8290.CD-18-0044

13. Kosorukov V.S., Baryshnikova M.A., Kosobokova E.N. et al. Identification of immunogenic mutant neoantigens in the genome of murine melanoma. Rossiysky bioterapevtichesky zhurnal = Russian Journal of Biotherapy 2019;18(3):23–30. (In Russ.). DOI: 10.17650/1726-9784-2019-18-3-23-30

14. Baryshnikova M.A., Rudakova A.A., Sokolova Z.A. et al. Evaluation of the antitumor efficacy of synthetic neoantigen peptides for the melanoma vaccine model Rossiysky bioterapevtichesky zhurnal = Russian Journal of Biotherapy 2019;18(4):76–81. (In Russ.). DOI: 10.17650/1726-9784-2019-18-4-76-81

15. Rudakova A.A., Baryshnikova M.A., Sokolova Z.A. et al. Evaluation of immunogenicity of synthetic neoantigen peptides for the melanoma vaccine model. Rossiyskiy bioterapevticheskiy zhurnal = Russian Journal of Biotherapy 2021;20(2):61–8. (In Russ.). DOI: 10.17650/1726-9784-2021-20-2-61-68

16. Stadler J.-C., Belloum Y., Deitert B. et al. Current and future clinical applications of ctDNA in immuno-oncology. Cancer Res 2022;82(3):349–58. DOI: 10.1158/0008-5472.CAN-21-1718

17. Tomayko M.M., Reynolds C.P. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24(3):148–54. DOI: 10.1007/BF00300234

18. Danciu C., Falamas A., Dehelean C. et al. A characterization of four B16 murine melanoma cell sublines molecular fingerprint and proliferation behavior. Cancer Cell Int 2013;13:75. DOI: 10.1186/1475-2867-13-75

19. Nicolson G.L., Brunson K.W., Fidler I.J. Specificity of arrest, survival, and growth of selected metastatic variant cell lines. Cancer Res 1978;38(11 Pt 2):4105–11. PMID: 359132

20. Nedosekin D.A., Sarimollaoglu M., Ye J.-H. et al. In vivo ultrafast photoacoustic flow cytometry of circulating human melanoma cells using near-infrared high-pulse rate lasers. Cytometry A 2011;79(10):825–33. DOI: 10.1002/cyto.a.21102

21. Herrera L.J., Raja S., Gooding W.E. et al. Quantitative analysis of circulating plasma DNA as a tumor marker in thoracic malignancies. Clin Chem 2005;51(1):113–8. DOI: 10.1373/clinchem.2004.039263

22. Sánchez-Herrero E., Serna-Blasco R., Robado de Lope L. et al. Circulating tumor DNA as a cancer biomarker: An overview of biological features and factors that may impact on ctDNA analysis. Front Oncol 2022;12:943253. DOI: 10.3389/fonc.2022.943253

23.