Stimulation of hepatocarcinogenesis by neutrophils upon induction of oncogenic kras expression in transgenic zebrafish

Background & aims: Chronic inflammation is a major etiological factor for hepatocellular carcinoma (HCC), but how immune cells respond in the initiation of hepatocarcinogenesis remains uncharacterized. This study aims to investigate the response and roles of neutrophils in early hepatocarcinogenesis. Methods: By inducible expression of oncogenic kras(V12) in hepatocytes in transgenic zebrafish combined with live imaging of neutrophils in transparent larvae, the response of neutrophils to oncogenic liver was characterized and their roles investigated by pharmaceutical and genetic manipulations. Results: We found a rapid recruitment of neutrophils to the liver upon induction of kras(V12) expression. Pharmaceutical stimulation of neutrophils resulted in further increases of neutrophils in oncogenic livers, liver size and tumor severity, while inhibition of neutrophils caused decreases of liver-associated neutrophils and liver size. Time-lapse video indicated that neutrophils had a stagnant migratory pattern meandering along the tumor edge but became relatively stationary upon entering the kras(V12)-expressing liver. Both oncogenic hepatocytes and tumor-associated neutrophils (TANs) were isolated via fluorescence-activated cell sorting. Molecular analyses indicated a pro-inflammatory microenvironment, as marked by increased tgfβ1a expression in kras(V12)-expressing hepatocytes and a loss of anti-tumor activities in TANs. Depletion of Tgf-β significantly reduced the number of TANs and the size of oncogenic liver. Conclusions: An inflammatory cue from oncogenic hepatocytes upon induction of kras(V12) expression causes a rapid recruitment of neutrophils to oncogenic liver and the neutrophils play a promoting role in early hepatocarcinogenesis.