2629 Background: The complex cell interactions within the tumor microenvironment (TME) have become a crucial point in cancer research. Yet, the cell interactions might not only depend on the frequency of immune cells, but also on the inter-individual distances as cells might interact via soluble factors and/or cell-cell contact. Accordingly, the mapping of TME has recently gained importance. The aim of this study is to investigate the alternations between galectin-9 (G9) and its natural immunosuppressive receptor, T cell immunoglobulin and mucin domain 3 (Tim3) in nasopharyngeal cancer (NPC). Methods: Using multiplexed quantitative immunofluorescence, we measured the levels of G9 and Tim3 in 95 NPC patients cancerous and 8 normal specimens in tissue microarray format. Cell densities and cell-to-cell distances were quantified. The interaction between G9-expressing tumor cell lines and T cells were also studied. Results: G9-expressing tumor cells were detected in all NPC cases and were significantly higher than normal tissue. Elevated G9 was associated with shorter overall survival (OS: 89% vs 70.5% at 7 years, p: 0.019). Incremental percentages of Tim3⁺ cells were shown in top 10% cases strongly positive for G9-expressing tumor cells. The number of Tim3⁺ cells was calculated at 15µm intervals from the nearest G9-expressing tumor cells, of which a significant difference of Tim3⁺ cells was observed at the 0-15µm distance from G9-expressing cell in cancerous compared to normal tissues. Epithelial short distances were associated with a unfavourable prognosis. Observed short distance were hypothesized to represent Tim3⁺ cells actively interacting with G9-expressing tumor cells. Accordingly, In vitro cocultured of G9-ovexpressing NPC cell lines induced Tim3 expression on T cells which suppressed the T-cell mediate cytotoxicity on tumor cells. Conclusions: Our findings indicate a specific preexisting profile of Tim3⁺ and G9-expressing tumor cells and demonstrated that Tim3⁺ cells were mainly found intratumorally within 15µm of a NPC cell. The relevance of Tim3⁺ and G9⁺ distances reflect a potential marker of their functional interaction. Our results could have important implications for clinical therapeutic strategies. Since high G9 expression have poorer OS, they would deserve a different therapeutic strategy.[Table: see text]