Esophageal adenocarcinoma cells do not traffic Fas Ligand to the cell surface: Evidence against the “Counterattack” hypothesis

Abstract

Introduction. The expression of Fas Ligand (FasL) in normal tissues is limited to activated lymphocytes, macrophages, and immune-privileged tissues such as the testis, eye, brain, and placenta. The finding that many tumors also express FasL spawned the “Counterattack” hypothesis, which suggests that FasL-expressing tumors evade immune surveillance by inducing apoptosis of infiltrating lymphocytes that abundantly express the Fas receptor. We hypothesized that EA cell lines express FasL but do not traffic it to the cell surface, making them incapable of inducing Fas-mediated apoptosis of immune cells. Methods. FasL-expressing Jurkat T cells (positive control), immortalized squamous esophagus cells (SE), immortalized Barrett’s esophagus (BE), and three esophageal adenocarcinoma (EA) cell lines were studied for FasL expression by immunoblotting. Subcellular localization was analyzed by immunostaining using laser-scanning confocal microscopy and FACScan analysis of cell-surface expression. Culture media were examined for soluble FasL by ELISA. Results. FasL expression was observed in all three EA cell lines and to a lesser degree in the SE cell line, but not in the immortalized BE cell line. Immunofluorescence of EA cells demonstrated that FasL protein was localized to the cytoplasm, predominantly in a perinuclear distribution. FACScan analysis confirmed the EA cell lines lack cell-surface expression, even after inhibition of matrix metalloproteinases. Trace amounts of FasL were detected in the media of all cell lines, but these levels did not differ from media supplemented with 10% fetal bovine serum alone (ANOVA). Conclusions. EA cells express FasL but do not traffic it to the cell surface. Thus, the function of FasL in EA is unlikely to be a “Counterattack” of immune cells. FasL expression in tumors may confer other advantages to the cancer cell.