S. Tohme1, H. Huang1, A. Al-Khafaji1, A. Tsung1 1University Of Pittsburgh,General Surgery,Pittsburgh, PA, USA
Introduction: Previous studies have shown that after liver ischemia-reperfusion (I/R), the growth of hepatic metastatic tumors increase; however, the mechanisms remain unclear. We have previously shown that during I/R neutrophils release Neutrophil Extracellular Traps (NETs), extracellular fibers composed of DNA and neutrophil proteins, which heighten the inflammatory response and subsequent liver injury. Some emerging data suggests that NETs may play a role in tumor progression, but the role of NETs in I/R-mediated acceleration of tumor growth is unknown. We hypothesize that NETs formed in response to liver I/R promote tumor growth and progression.
Methods: In-vitro, neutrophils were harvested from mice bone marrow. Neutrophils were treated with PMA, a well-known stimulator of NET formation, for 4hrs. Media was collected and co-cultured with mc38 cancer cells. MTT assays, Western blots, invasion and migration assays were used for analysis. In-vivo, colorectal liver metastases were induced in C57BL/6 mice by spleen injection of mc38 cells. Animals were then subjected to partial liver I/R vs. sham surgery followed by splenectomy with or without daily DNase I injections, a known inhibitor of NETs. The livers were harvested 3 weeks later for analysis.
Results: In vitro, there was a significant increase in proliferation of mc38 cells co-cultured with stimulated neutrophil media compared to untreated mc38 cells by MTT assay. This surge in proliferation was significantly decreased when DNaseI was added. Similarly, there a was significant increase in the invasion and migration of mc38 cells treated with stimulated media compared to control or addition of DNaseI. By using western blot analysis, the addition of neutrophil media resulted in the activation and phosphorylation of STAT3, a protumorigenic transcription factor, and p38, p65 and JNK, mitogen activator protein kinases implicated in tumor growth and development under stressful conditions. The activation of these proteins was significantly decreased with the addition of DNaseI. In vivo, Mice in the I/R group had grossly significantly more tumor growth and higher liver/body weight when compared to mice in the sham group. Addition of DNaseI to mice receiving I/R resulted in a significant decrease in tumor growth. Histologically, the tumors from the I/R + DNaseI group has less proliferation by Ki67 staining compared to the I/R group. There was a similar decrease in the activation of the MAP kinases from the tumor tissue obtained from the I/R + DNase1 mice compared to the I/R group. There was no difference in tumor growth between mice in the sham groups with or without DNaseI.
Conclusions: Liver I/R is a strong stimulus for metastatic tumor growth. NETs formed during I/R contributes to tumor growth by activating protumorigenic signaling pathways. DNaseI may represent a novel therapy for targeting NET-mediated tumor growth during liver I/R.