The detachment of tumor cells from extracellular matrix and survival under anchorage-independence were recognized as the initial step of tumor metastasis. Previously we had demonstrated that anchorage-independence altered gene expressions and showed characteristics of cell invasiveness loss, enhanced chemosensitivity, and enhanced subcutaneous tumor formation. However, whether it affected histological phenotypes in tumor tissues remained unclear.

Melanoma metastases were generated in nude mice using adherent or suspended melanoma cells. Examination of melanoma metastases revealed histological features of extensive vascular structures in adherent cell-derived tumors, while not seen in suspended cell-derived tumors. Quantitative proteomic analysis at adherent, suspended, and re-attached melanoma cells suggested that aminopeptidase N was potentially downregulated upon cell suspension or reattachment. Downregulation of aminopeptidase N by gene-specific shRNAs showed reduced cell invasiveness and enhanced subcutaneous tumor formation that was consistent with previous observations. Experiments by suppression or overexpression of aminopeptidase N expression demonstrated that aminopeptidase N regulated syndecan-1 and integrin β4 expression through PKCδ pathway. Histological analysis at melanoma metastases further suggested that CD31+/aminopeptidase N+/syndecan-1+/integrin β4+ phenotypes were associated with vascular structures.

In summary, we suggested the expression axis of aminopeptidase N/syndecan-1/integrin β4 in melanoma cells was suppressed by detachment stress, which diminished vascular phenotypes of melanoma metastases.