Induction of apoptosis in the centre of multicellular tumour spheroids of colorectal adenocarcinomas - involvement of CD95 pathway and differentiation

Multicellular tumour spheroids (MCTS) are three-dimensional cell culture sy stems which are widely used in cancer research. They are characterized by a n outer zone of proliferating cells, an inner region of differentiating qui escent cells and an area of so-called necrotic cell death in their centre. The exact cause of this cell death, a controversy for many years, was the a im of the present study. Our data show that cell death in the centre of MCT S of three colorectal adenocarcinoma cell lines (HRT-18, HT-29 and CX-2) wa s induced by apoptosis. Apoptotic cells were initially distributed at rando m but accumulated very quickly in the quiescent and central area at day 4-5 , suggesting a time- rather than size-dependent synchronization of apoptosi s parallel to the formation of the proliferation gradient in MCTS. To study mechanisms inducing apoptosis, the Fas-pathway was investigated. A cell-ce ll contact-dependent expression of CD95 was found in all MCTS. Fast was not detected in monolayer cultures, but was expressed in spheroids of HRT-18 a nd CX-2. We found that TNF alpha and TGF beta 1 activated the CD95 pathway in all three cell lines. Since both TNF-cr and TGF-P are known to be induci ble by hypoxia in a variety of cell types, we suggest that these hypoxia-in duced factors sensitize the CD95 pathway in the quiescent area of MCTS. Fur thermore, a loss of the heat shock proteins 27, 32, 60, 73 and 90 was obser ved in the quiescent area of spheroids. This suggests that tumour cell diff erentiation in the inner region of MCTS may be an additional factor inducin g apoptosis.