Objective: The objective was to evaluate the enhancement of human peritoneal macrophage cytotoxic in vitro activity by the addition of interleukin-2 (IL-2) to the standard interferon gama (IFNgamma) and lipopolysaccharide (LPS) activation procedure used for cellular adoptive immunotherapy in a human ovarian cancer system. This cytotoxic effect of these activated macrophages was tested on cells from ovarian cancers of various stages, histology type, and grade, both prior to chemotherapy and at recurrence, in ovarian carcinoma cells lines and normal cells. Increased activation of the macrophage may make it a better candidate for intraperitoneal cellular adoptive immunotherapy as a component of ovarian cancer therapy. This was not a study of the mechanism of macrophage killing.
Methods: Ascites specimens were collected from 24 ovarian cancer patients at the time of surgery or by paracentesis. The mononuclear cell fraction was isolated by discontinuous density gradient centrifugation and used as a cellular source of peritoneal macrophages (PMs) and primary cultured ovarian cancer cells. PMs were separated by 1-h adhesion followed by intensive washing to remove floating cells. The floating cells were cultured for 24 h which left the cancer cells attached after unattached cells were removed by washing. These cells formed a monolayer of cancer cells, which could be subcultured in 22 patients. The cells from the third to fifth passages were used as target cells without coculture with other cells. PMs were identified by latex ingestion, and their purity after isolation by adhesion culture was tested by flow cytometry and immunofluorescence. PMs were activated by culturing in the presence of IFNgamma, with or without IL-2, for 18 h followed by the addition of LPS 6 h prior to use as effector cells in cytotoxicity assays. Ovarian cancer cells of both established cell lines and primary cultures were labeled with (51)Cr and utilized as target cells to quantitatively measure PM-mediated cytotoxicity. Ovarian cancer cells were also cocultured with PMs for morphologic observations to provide supporting evidence to the cytotoxicity assays.
Results: IL-2 enhances the cytotoxicity of the standard IFNgamma/LPS macrophage activation in this system. Peritoneal macrophages so activated are cytotoxic to autologous and allogenic primary cultured ovarian tumors and to ovarian carcinoma cell lines. The macrophages are cytotoxic to cells both prior to treatment and at recurrence, but the data from the few recurrent patients did reach statistical significance. This cytotoxicity is not MHC associated. Normal cells are minimally affected.
Conclusions: IL-2 augmented the standard IFNgamma/LPS method of activating peritoneal macrophage cell killing of human ovarian cancer cells in this in vitro system. The cell killing occurred with autologous and allogenic tumor cells from patients with primary and possibly recurrent tumors. Activated PMs minimally affected the normal cells tested. This enhanced activation may improve the disappointing results of previous adoptive cellular immunotherapy human trials and should be considered for ovarian cancer clinical trials.
Copyright 1999 Academic Press.