Characterization of the release and biological significance of cell-free DNA from breast cancer cell lines

Abstract

In breast cancer, cell-free DNA (cfDNA) has been proven to be a diagnostic and prognostic biomarker. However, there have been few studies on the origin and biological significance of cfDNA. In this study, we assessed the release pattern of cfDNA from breast cancer cell lines under different culture conditions and investigated the biological significance of cfDNA. The cfDNA concentration increased rapidly (6 h) after passage, decreased gradually, and was then maintained at a relatively stable level after 24 h. In addition, the cfDNA concentration did not correlate with the amount of apoptotic and necrotic cells. Interestingly, if more cells were in the G1 phase, more cfDNA was detected (p < 0.01) and the cfDNA concentration correlated positively with the percent of cells in the G1 phase (p < 0.05). We observed that cells could release cfDNA actively, but not exclusively, via exosomes. Furthermore, we showed that cfDNA could stimulate hormone receptor-positive breast cancer cell proliferation by activating the TLR9-NF-κB-cyclin D1 pathway. In conclusion, cfDNA is released from breast cancer mainly by active secretion, and cfDNA could stimulate proliferation of breast cancer cells.

Keywords: biological significance; breast cancer; cell-free DNA; circulating tumor DNA.

Figure 1. CfDNA concentration in the supernatant of MCF-10A, T47-D, and MDA-MB-231 cell lines under normal culture conditions

The number at the top of each bar represents the relative concentration at each time point. Total concentration: the cfDNA concentration in the supernatant of breast cancer cells. Normalized concentration: the cfDNA concentration was normalized in terms of the amount of viable cells.

Figure 2. Concentration of cfDNA from breast cancer cells with different levels of apoptosis and necrosis

(A) Scatter diagram of the flow cytometry of cells treated with different doses of the apoptotic inducer; (B–D) histograms of the apoptosis and necrosis ratio of MCF-10A, T47-D, and MDA-MB-231 cells; (E) CfDNA concentration of cell lines treated with different doses of the apoptotic inducer.

Figure 3. Relationship of cfDNA concentration and cell-cycle

(A–D) Cell-cycle analysis of three cell lines treated with different doses of cell-cycle inhibitor; (E) CfDNA concentration of cell lines treated with different doses of cell-cycle inhibitor.

Figure 4. CfDNA concentration of exosomes and the corresponding culture supernatant

(A) Cell-cycle analysis of cells cultured with or without FBS; (B) CD9 expression of exosomes extracted from the supernatant with or without FBS; (C) CfDNA concentration of total supernatant (all), exosomes (exosomes (+)) and supernatant without exosomes (exosomes (−)). +: culture with FBS; —: culture without FBS.

Figure 5. The promotion of T47-D and MCF-7 cells proliferation by cfDNA

(A) Clone formation assay of cells treated with cfDNA; (B) CCK-8 assay of cells treated with cfDNA; (C) cell-cycle analysis of cells treated with cfDNA.

Figure 6. Protein expression levels of TLR9-NF-κB pathway members after the cells were treated with cfDNA

(A) Protein expression levels of TLR9, p-P65, and cyclin D1 in T47-D and MCF-7 cells after treatment with cfDNA, CQ, cfDNA+CQ, DNase I, DNaseI+cfDNA; (B) protein expression levels of IKK α + β, IKK α, ERα, and p- ERα in T47-D and MCF-7 cells after treated with cfDNA, CQ, cfDNA+CQ, DNase I, DNase I+cfDNA. CQ: chloroquine; p-P65: phosphorylated P65; p-ERα; phosphorylated Erα.