ROCK signaling is involved in the entosis of both nonepithelial and epithelial tumors, whereas N‑cadherin is involved in the entosis of nonepithelial tumors

Entosis is a cell-in-cell phenomenon wherein cells detaching from the extracellular matrix are internalized by a neighboring cell. The present study examined whether entosis, which is observed in epithelial cells, also occurs in nonepithelial cells. The present study used the MCF-7 breast cancer cell line as a positive control for entosis and compared this with three representative sarcoma cell lines (RD, HT1080 and ICH-ERMS-1). The formation of cell-in-cell structures was induced by culturing cells in adherent and nonadherent conditions. Cell lines that formed the cell-in-cell structures were cultured in nonadherent conditions with and without Rho-associated coiled-coil containing protein kinase (ROCK) inhibition, and the cell-in-cell structures were evaluated in slides prepared from cell blocks. It was examined whether ROCK inhibition blocked the formation of cell-in-cell structures, and the expression levels of specific cadherins associated with entosis were determined using quantitative PCR. The proportion of cells with cell-in-cell structures was significantly higher in nonadherent conditions in both MCF-7 (P=0.0297) and RD (P=0.0098) cells, whereas few cell-in-cell structures were observed in both adherent and nonadherent conditions in HT1080 and ICH-ERMS-1 cells. Under nonadherent conditions, ROCK inhibition significantly reduced the proportion of cells with cell-in-cell structures in MCF-7 (P=0.0021) and RD (P=0.0407) cells. Based on quantitative PCR, among the five cadherin genes, the E-cadherin expression level was the lowest in MCF-7 cells (ΔCt, 2.6) and the N-cadherin expression level was lowest in RD cells (ΔCt, 4.8). By contrast, the N-cadherin expression levels were higher in HT1080 (ΔCt, 11.0) and ICH-ERMS-1 (ΔCt, 8.8) cells. These results suggested that the cell-in-cell phenomenon observed in RD cells is an entotic process based on its emergence in nonadherent culture conditions and the involvement of ROCK signaling. Entosis observed in RD cells was mediated via N-cadherin and not E-cadherin.