Potentiatied antitumor effectiveness of combined chemo-immunotherapy with interleukin-12 and 5-fluorouracil of L1210 leukemia in vivo.

In this study we investigated the efficacy of a combination of IL-12 and 5-FU, a chemotherapeutic exerting several immunomodulatory effects, in murine L1210 leukemia. Mice inoculated with 1 x 10(5) leukemia cells were treated with a single dose of 5-FU (50 mg/kg) and seven daily doses of IL-12 (100 ng/dose), and were observed for survival. Treatment with IL-12 or 5-FU given alone produced moderate anti-leukemic effects. However, combination of both drugs resulted in a significant prolongation of mouse survival time. Importantly, there were 70% of long-term (>60 days) survivors among mice treated with both agents simultaneously. Moreover, we observed 100% of long-term survivors when mice were treated with a minimally increased dose of IL-12 (170 ng) in combination with 5-FU (50 mg/kg). The antileukemic effects were completely abrogated in scid/scid mice and in mice depleted of peritoneal macrophages and significantly decreased after administration of anti-CD3+, anti-CD4+ or anti-CD8+ monoclonal antibodies. Administration of anti-NK1.1 antibodies did not decrease the antileukemic effects indicating that NK cells are not important effectors of this treatment regimen. Collectively, these results indicate that the combination of IL-12 and 5-FU is inducing strong antileukemic responses that are dependent on the presence and activity of macrophages and T lymphocytes and warrant further studies of combined chemo-immunotherapy with IL-12.

Direct stimulation of macrophages by IL-12 and IL-18--a bridge too far?

A novel pathway of autocrine macrophage activation based on a positive feedback loop involving interleukin (IL)-12, IL-18 and IFN-gamma has recently been suggested. However, the macrophage isolation technique employed to describe the above phenomenon does not allow obtaining a pure population of macrophages casting some doubt to its existence. In the present study, we show that even minor contamination with lymphoid cells of a pure population of macrophage-like cells (Raw 264.7) results in a marked production of nitric oxide after stimulation with both IL-12 and IL-18. Neither macrophage-like cells nor lymphoid cells were capable of secreting high amounts of nitric oxide after stimulation with IL-12 and/or IL-18. Based on these observations we hypothesize that proposed autocrine feedback loop of macrophage activation is rather paracrine in nature and involves direct stimulation of residual lymphoid cells to secrete IFN-gamma that is then capable of activating macrophages.

Potentiatied anti-tumor effectiveness of combined therapy with interleukin-12 and mitoxantrone of L1210 leukemia in vivo.

In previous studies we have shown that combined chemo-immunotherapy of L1210 leukemia with IL-12 and doxorubicin results in striking anti-tumor effects producing 100% of long-term survivors. In this study we investigated the efficacy of a combination of IL-12 and mitoxantrone in murine L1210 leukemia. Mice inoculated with 1x105 leukemia cells were treated with a single dose of mitoxantrone and seven daily doses of IL-12, and were daily observed for survival. Treatment with IL-12 or mitoxantrone given alone produced moderate anti-leukemic effects. However, combination of both drugs resulted in a significant prolongation of mouse survival time. Importantly, there were almost 50% of long-term (>60 days) survivors among the mice treated with both agents. This therapeutic effect was completely abrogated by sub-lethal, whole-body X-irradiation, and significantly reduced after macrophage depletion.

Interleukin 12 and indomethacin exert a synergistic, angiogenesis-dependent antitumor activity in mice.

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence and mortality from colorectal cancer. It has recently been demonstrated that these drugs are capable of suppressing the production of pro-angiogenic factors from tumor cells. The mechanisms of antitumor action of interleukin 12 include the enforced secretion of anti-angiogenic factors and stimulation of antitumor immunity. Therefore, we hypothesized that the combination of a model nonsteroidal anti-inflammatory drug--indomethacin and interleukin 12--would result in enhanced angiogenesis-dependent antitumor effects against a colon-26 carcinoma cells transplanted into syngeneic mice. As expected the combined administration of both agents simultaneously resulted in a strengthened antitumor activity that was manifested as a retardation of tumor growth and prolongation of mouse survival. Importantly some mice were completely cured after the combined treatment. As administration of interleukin 12 and indomethacin resulted in enhanced inhibition of angiogenesis it seems possible that prevention of new blood vessel formation is one of the mechanisms responsible for the observed antitumor effects.

Potentiation of the anti-tumour effects of Photofrin-based photodynamic therapy by localized treatment with G-CSF.

Photofrin-based photodynamic therapy (PDT) has recently been approved for palliative and curative purposes in cancer patients. It has been demonstrated that neutrophils are indispensable for its anti-tumour effectiveness. We decided to evaluate the extent of the anti-tumour effectiveness of PDT combined with administration of granulocyte colony-stimulating factor (G-CSF) as well as the influence of Photofrin and G-CSF on the myelopoiesis and functional activity of neutrophils in mice. An intensive treatment with G-CSF significantly potentiated anti-tumour effectiveness of Photofrin-based PDT resulting in a reduction of tumour growth and prolongation of the survival time of mice bearing two different tumours: colon-26 and Lewis lung carcinoma. Moreover, 33% of C-26-bearing mice were completely cured of their tumours after combined therapy and developed a specific and long-lasting immunity. The tumours treated with both agents contained more infiltrating neutrophils and apoptotic cells then tumours treated with either G-CSF or PDT only. Importantly, simultaneous administration of Photofrin and G-CSF stimulated bone marrow and spleen myelopoiesis that resulted in an increased number of neutrophils demonstrating functional characteristics of activation. Potentiated anti-tumour effects of Photofrin-based PDT combined with G-CSF observed in two murine tumour models suggest that clinical trials using this tumour therapy protocol would be worth pursuing.