J. E. McDade1, J. Naqvi2, J. Lee2, D. S. Pisetsky3,4, B. A. Sullenger2, T. V. Brennan2, R. R. White2 1Duke University Medical Center,Durham, NC, USA 2Duke University Medical Center,Department Of Surgery,Durham, NC, USA 3Duke University Medical Center,Department Of Rheumatology And Immunology,Durham, NC, USA 4Duke University Medical Center,Department Of Medicine,Durham, NC, USA
Introduction: Circulating cell-free DNA (cfDNA) is known to correlate with tumor stage in several different cancer types. Levels of cfDNA are increased in the serum of pancreatic cancer (PC) patients, and increasing evidence shows cfDNA may play a role in mediating disease progression. Degradation of cfDNA using DNase I reduces pancreatic cancer cell invasion in vitro and reduces metastasis in a PC mouse model. These data indicate that cfDNA is more than a biomarker and may also be a target for therapy. For this purpose, we propose to use nucleic acid binding polymers (NABPs) as a more clinically feasible approach to reverse the effects of cfDNA. Our lab has previously demonstrated that NABPs can bind and reverse the effects of cfDNA in vitro and improve outcomes in various inflammatory conditions. Our hypothesis is that cfDNA will increase PC cell invasion in vitro, and that NABPs can ‘scavenge’ cfDNA and block this effect.
Methods: Human PC cell lines Panc-1 and BxPC-3 were used for the following experiments. Cells were seeded in serum-free media onto a Matrigel-coated Transwell membrane with 8μm pores and allowed to invade for 24hr. Invaded cells were fixed, stained with 5% crystal violet, and the average number of invaded cells per image was counted. We examined the effects that various cfDNA sources had on invasion and investigated the co-administration of PAMAM-G3, a commercially available NABP, to assess its potential as a therapeutic agent.
Results: CpG ODN 2006 (4μM), a synthetic unmethylated DNA oligonucleotide, potently stimulates Panc-1 and BxPC-3 invasion. Sources of DNA in the serum of PC patients include direct secretion from immune cells, or release from dying cancer cells. To emulate these settings, we investigated naturally occurring sources of cfDNA and found that conditioned media (CM) containing high levels of cfDNA released from irradiated PC cells stimulate BxPC-3 invasion. PAMAM-G3 (20μg/mL) blocks the effects of both CpG and endogenous cfDNA from CM and reduces PC cell invasion back to baseline levels (Fig A). In a separate experiment, purified mitochondrial DNA (mtDNA), which contains a higher proportion of unmethylated CpG sequences than genomic DNA, also stimulates PC cell invasion at 20μg/mL and may be the component of cfDNA that is mediating this effect (Fig B).
Conclusion: CfDNA-containing media and purified mtDNA stimulate PC cell invasion in vitro, and the effects of cfDNA can be blocked by PAMAM-G3. These data provide evidence that cfDNA may be an important mediator of disease progression in PC and suggest a potential therapeutic application for NABPs.
