Tumour-induced apoptosis in human mesothelial cells: a mechanism of peritoneal invasion by Fas Ligand/Fas interaction

Abstract

Gastrointestinal carcinomas frequently disseminate within the abdominal cavity to form secondary peritoneal metastases. Invasion of the peritoneal mesothelium is fundamental to this process, yet the underlying invasive mechanisms remain unclear. Preliminary in vitro work suggested that tumour cells can induce mesothelial apoptosis, representing a novel mechanism of peritoneal invasion. We examined the role of tumour cell-induced mesothelial apoptosis and explored the role of the death ligand/receptor system, Fas Ligand/Fas, as mediators of the apoptotic process. Cultured human mesothelial cells were used to establish in vitro co-culture models with the SW480 colonic cancer cell line. Tumour-induced mesothelial apoptosis was confirmed by phase-contrast microscopy and apoptotic detection assays. Human mesothelial cells and SW480 tumour cells constitutively expressed Fas and Fas Ligand mRNA and protein as determined by RT-PCR and confocal fluorescent microscopy. Stimulation of human mesothelial cells with anti-Fas monoclonal antibody or crosslinked soluble Fas Ligand-induced apoptosis, confirming the functional status of the Fas receptor. Pretreatment of SW480 cells with a blocking recombinant anti-Fas Ligand monoclonal antibody significantly reduced mesothelial apoptosis, indicating that tumour-induced mesothelial apoptosis may, in part, be mediated via a Fas-dependent mechanism. This represents a novel mechanism of mesothelial invasion and offers several new targets for therapeutic intervention.

Figure 1

Mesothelial-SW480 co-cultures. The tumour cells, T, induce apoptosis in adjacent mesothelial cells, M. Mesothelial DNA fragmentation is apparent as brown nuclear staining. Cell shrinkage and characteristic membrane blebbing are apparent in (A) (Tumour cells unstained in this experiment). (B–D), Dual staining of mesothelial-SW480 co-cultures. Tumour cells (T) are identified by their position in the focal plane and by their positive membrane staining (red) for BerEp4 epithelial antigen. Adjacent mesothelial cells (M) show apoptotic changes with nuclear fragmentation (brown). Bar, 2.5 μm.

Figure 2

Assessment of the functionality of human mesothelial Fas. Mesothelial monolayers were incubated with an agonistic anti-Fas mAb or stimulating crosslinked sFasL (dark columns). Controls were untreated mesothelial monolayers (light columns). Mesothelial apoptosis was detected using a TUNEL assay. Results are expressed as mean AI of triplicate experiments±s.d. ^*P<0.05, Mann–Whitney U-test.

Figure 3

Quantitative co-culture apoptosis assays. SW480 cells were pretreated with a FasL inhibiting recombinant protein rhFas:Fc prior to addition to mesothelial monolayers as co-cultures (CC). Monolayers (M) and SW480 tumour cells (SW) were cultured alone as controls. ± denotes pretreatment of cultures with recombinant protein. All co-culture experiments were terminated at 8 h. Mesothelial cell death is expressed as mean AI of triplicate experiments±s.d. ^*P<0.05, Mann–Whitney U-test.

Figure 4

FasL/Fas interaction in tumour-mediated mesothelial apoptosis. Dual confocal fluorescent microscopy of co-cultures with anti-FasL and anti-Fas antibodies. FasL and Fas were visualised with a TRITC and FITC secondary antibody respectively. Mesothelial cells demonstrate homogenous cell surface expression of Fas (A) and undergo cytoplasmic retraction following tumour cell interaction (B). Tumour cell FasL is polarised at areas of mesothelial contact (C, broken arrow). FasL is not constitutively expressed by mesothelial cells but is induced by the presence of adherent tumour cells (C, arrows). FasL channel only (D). M, mesothelial cell. T, SW480 tumour cell. (A) Bar 20 μm; (B) bar 20 μm; (C) bar 5 μm; and (D) bar 10 μm.