INTERACTIONS OF LYMPHOKINE-ACTIVATED KILLER-CELLS AND A549-LUNG-CARCINOMA NODULES MAINTAINED IN 3-DIMENSIONAL CULTURE

Lymphokine-activated killer (LAK) cells were cocultured in the presenc e of interleukin-2 (IL-2), with pulmonary surfactant secreting A549 lu ng carcinoma nodules and maintained in continuous three-dimensional cu lture for 2-6 days in an attempt to test the response of tumor cells w hich produce LAK cell inhibitory substances. The A549 nodules secrete mucus which envelops them. This mucus is also secreted inside pseudoal veolar structures characteristic of these nodules. The mucus contains the pulmonary surfactant and sialomucins, both LAK cell inhibitory sub stances. The spontaneous infiltration into the neoplastic tissue and t he membrane contacts established between the two cell types were studi ed by means of histological, immunohistochemical and electron-microsco pic methods. Free-floating LAK cells were allowed to sediment and adhe re freely to the nodule surface. The cytostatic and cytolytic effects of LAK cells were tested using thymidine incorporation into DNA and no w cytometry. Despite the presence of a mucus envelope, LAK cells adher ed to the A549 nodule surface and penetrated spontaneously into them i n the presence of IL-2: they settled mainly in the pseudoalveolar stru ctures where they became apoptotic. According to electron-microscopic observations performed on the second day of coculture, the LAM cells, which remained between the cancer cells, established mostly pinpoint c ontacts with the carcinoma cells, forming cytoplasmic fusions. These f usions indicate the induction of pores in both the cancer cell and the LAK cell membranes. Electronmicroscopic observation also displayed LA K-cell-associated apoptotic and necrotic carcinoma cells. However, al this stage of the coculture (day 2), the DNA synthesis rate of the A54 9 nodules still remained unchanged; it diminished by approximately 3 t imes on day 4 and almost stopped on tay 6: nodule disintegration was t hen complete. In the free-floating LAK cell component of the coculture s, DNA synthesis was already strongly inhibited (26 x)by the second da y. Nevertheless, their cytolytic effect remained unaltered, as was tes ted on A549 monolayer cells. The presence of tumor necrosis factor (TN F) in the coculture supernatant has been demonstrated, and when cocult uring took place in the presence of monoclonal TNF antibody, nodule pr oliferation was significantly enhanced (up to 145%). Our results indic ate that, despite the presence of pulmonary surfactant and sialomucins containing mucus, LAK cells were capable of killing lung carcinoma ce lls in three-dimensional culture at an early stage of coculture (day 2 ) by direct cell-to-cell contact. Total nodule disintegration, however , was complete much later (on day 6), and taking into account the low amount of LAK cells in the cancer tissue, this seemed to be the result of an indirect effect implying, in particular, the presence of solubl e TNF.