A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by tumor necrosis factor alpha (TNF-alpha) and CD8+ T cells were observed with tumor regression. Treatment-related adverse events were limited to mild to moderate pain at the injection site; clinically significant toxicities were not encountered. Transient but clear reductions of tumor sizes were observed at the injected sites in four of nine cases, and at noninjected distant sites in one melanoma patient. Hemorrhagic necrosis of tumors was observed in two melanoma patients. These data indicate that gene therapy by peritumoral injection of IL-12-producing autologous fibroblasts is feasible, and promising in patients with advanced cancer.

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Local production of reactive oxygen intermediates, e.g., superoxide anion, by tumor promoter-stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two-stage chemical-induced carcinogenesis in murine skin. In the studies reported herein, peritoneal MPs elicited from phorbol-ester-sensitive SENCAR mice demonstrated a time- and dose-dependent release of superoxide anion (4-6 nmol/10(6) cells) when stimulated by 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro; MP superoxide response was significantly inhibited (50-70%) by preincubation with 40 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a protein-kinase inhibitor. Alternatively, TPA-stimulated MPs derived from relatively resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain-dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter previously shown to act independently of protein kinase C (PKC). TG-stimulated SENCAR MPs released a significant amount of superoxide (2-3 nmol/10(6) cells) that was not inhibited by H-7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with 100 microM dibromoacetophenone, an inhibitor of phospholipase A2, completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 50 microM 1-oleoyl-2-acetylglycerol, a diacylglycerol analogue and PKC activator, also induced a significant amount of superoxide from SENCAR MPs only. In parallel with the superoxide findings, TPA and TG stimulated significantly greater [3H]arachidonic acid release from prelabeled SENCAR MPs (a 32% and 48% increase, respectively, over unstimulated controls) relative to MPs from B6C3F1 mice. Two-dimensional gel-electrophoretic analysis indicated that TPA-induced phosphorylation of a 47-kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig and human polymorphonuclear leukocytes) occurred in MPs from both SENCAR and B6C3F1 mice. Therefore, arachidonic acid production may be a common biochemical pathway by which phorbol-ester--and non-phorbol-ester--type tumor promoters activate MPs in SENCAR mice; such a response may be "permissive" for additive (or synergistic) interactions with PKC-driven signal pathways.

SSIN mice are considerably more sensitive to the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) in two-stage skin carcinogenesis protocols than are most other strains and stocks of mice. Experiments were performed to determine whether there was an immunological basis for this sensitivity. SSIN mice were haplotyped and found to be H-2q. T cells represented approximately 31% of the splenic cellularity of non-treated SSIN mice, but approximately 44% in BALB/c, C57BL/6, B6C3F1 and SENCAR mice. Splenic CD4+/CD8+ T cell ratios were approximately 4.2, 2.9, 2.4, 1.8 and 1.7 in SSIN, SENCAR, BALB/c, B6C3F1 and C57BL/6 mice, respectively. The unusually high ratio in SSIN spleens was the consequence of reductions in CD8+ T cells. The ratio of CD4+/CD8+ T cells in SSIN thymocytes was similiar to that measured in the spleen. The splenic cytotoxic T lymphocyte (CTL) activities of the various murine strains inversely correlated with their splenic CD4+/CD8+ ratios and their sensitivities in two-stage skin carcinogenesis protocols. Repeated in vivo topical treatment of SSIN mice with TPA caused significant decreases in splenic T cell contents, but affected neither the splenic CD4+/CD8+ T cell ratio nor the development of a CTL response upon allogeneic tumor challenge. SSIN mice also had very low splenic natural killer cell activities. Furthermore, relative to the other strains of mice, SSIN mice were poor responders upon alloantigen challenge in mixed lymphocyte response assays. These findings demonstrate that SSIN mice differ markedly from other murine stocks and strains in their splenic lymphocyte composition and in their abilities to mount some MHC-restricted and non-restricted immunosurveillance processes.