Simple SummaryThe crucial role of tumor-associated macrophages (TAMs) in the disease progression of various types of tumors, including esophageal squamous cell carcinoma (ESCC), has been well established. To investigate the close interplay between cancer cells and TAMs, we previously established a direct co-culture system. Our aim was to identify useful molecular markers whose expression is regulated by interactions with cancer cells and TAMs, to predict patient prognoses, or to gain a deeper understanding of the ESCC mechanism. Our method revealed that direct contact with macrophages stimulated matrix metalloproteinase 9 (MMP9) production in ESCC cells, and that MMP9 played an integral role in cancer cell migration and invasion in vitro. Moreover, MMP9 expression in ESCC cells was significantly correlated with high infiltration of TAMs and poor clinical outcomes in immunohistochemical analysis. Our study suggests the potential for TAMs or MMP9 as important prognostic markers or therapeutic ESCC targets.AbstractTumor-associated macrophages (TAMs) contribute to disease progression in various cancers, including esophageal squamous cell carcinoma (ESCC). We have previously used an indirect co-culture system between ESCC cell lines and macrophages to analyze their interactions. Recently, we established a direct co-culture system to closely simulate actual ESCC cell-TAM contact. We found that matrix metalloproteinase 9 (MMP9) was induced in ESCC cells by direct co-culture with TAMs, not by indirect co-culture. MMP9 was associated with ESCC cell migration and invasion, and its expression was controlled by the Stat3 signaling pathway in vitro. Immunohistochemical analyses revealed that MMP9 expression in cancer cells at the invasive front (“cancer cell MMP9”) was related to high infiltration of CD204 positive M2-like TAMs (p< 0.001) and was associated with worse overall and disease-free survival of patients (p= 0.036 andp= 0.038, respectively). Furthermore, cancer cell MMP9 was an independent prognostic factor for disease-free survival. Notably, MMP9 expression in cancer stroma was not associated with any clinicopathological factors or patient prognoses. Our results suggest that close interaction with TAMs infiltrating in cancer stroma or cancer nests induces MMP9 expression in ESCC cells, equipping them with more malignant features.