TNF-alpha promotes progression of peritoneal metastasis as demonstrated using a green fluorescence protein (GFP)-tagged human gastric cancer cell line.

The mechanisms underlying progression of peritoneal metastasis by gastric cancer after micrometastasis formation remain unclear. In the present study, we investigated metastasis to the abdominal wall peritoneum, one of the major features of peritoneal spread, using a human gastric cancer cell line (GCIY-EGFP) tagged with the green fluorescence protein gene (GFP). This model allows sensitive, specific and sequential observation of metastasis development from the initial deposits to peritoneal carcinomatosis at the end stage. In the initial phase, GCIY-EGFP cells could form micrometastasis selectively on the omentum and mesenterium in a milky spot-dependent manner, but not on abdominal wall peritoneum lacking milky spots until the late stages. In vitro analysis using primary mesothelial cells revealed addition of TNF-alpha to decrease their stress fibers, leading to morphological change followed by exposure of the submesothelial extracellular matrix (ECM) in intercellular gaps. Such TNF-alpha pretreatment was found to enhance attachment of tumor cells to the mesothelial monolayer. When tumor cells were injected into the peritoneal cavity of TNF-alpha pretreated mice, they could metastasize to the abdominal wall peritoneum from the very early stages, resulting in accelerated accumulation of ascites than in TNF-alpha non-pretreatment controls. RT-PCR analysis revealed that tumor cells express cytokines and chemokines, including TNF-alpha. Furthermore, TNF-alpha treatment results in up-regulation of expression of monocyte chemoattractant protein-1 (MCP-1) and IL-8 by mesothelial cells and of TNF-alpha itself by inflammatory leukocytes in the peritoneal cavity. These results suggest that metastasis to the abdominal wall peritoneum occurs as a second step from the first omental metastasis in a milky spot-independent manner and that TNF-alpha derived from tumor cells, mesothelial cells and inflammatory leukocytes in the peritoneal cavity may be involved in the progression of peritoneal metastasis.