Squamous cell carcinoma of the external auditory canal (SCC-EAC) is rare and has a poor prognosis. SCC-EAC cases with high-grade tumor budding (TB) or poorly differentiated clusters (PDCs) are associated with shorter survival than those with low-grade TB or PDCs. Emmprin is a protein expressed in tumor cells that stimulates the production of matrix metalloproteinase (MMP)-2 by stromal fibroblasts to facilitate tumor invasion. Recently, we reported that emmprin forms a complex with CD73 to regulate MMP-2 production from fibroblasts in vitro. Here, we examined the association of emmprin and CD73 expression with TB or PDCs as well as with survival in 34 biopsy specimens of SCC-EAC patients. High tumoral emmprin expression was associated with high-grade TB, while high stromal CD73 expression was associated with high-grade PDCs. Furthermore, concurrent elevated expression of tumoral emmprin and stromal CD73 was determined to be an independent poor prognostic factor. In immunoprecipitation analyses, complex formation between emmprin and CD73 was demonstrated in vitro. MMP-2 production from fibroblasts was more abundant when co-cultured with tumor cells than from fibroblasts cultured alone. Furthermore, MMP-2 production was reduced by the transfection of CD73 siRNA in fibroblasts co-cultured with tumor cells. The colocalization of emmprin and CD73 was enhanced in not only the peripheral cells of the tumor cell clusters that interact with fibroblasts but also in the cells of intra-tumor clusters. Overall, this study provides novel insights into the roles of emmprin, CD73, and MMP-2 in tumor invasiveness.This article is protected by copyright. All rights reserved.