A Preclinical Model of Ionizing Radiation and TNF-alpha Gene Therapy for Cancer

Long term control of high-grade brain tumors is rarely achieved with current therapeutic regimens. This study sought to determine if pretreatment with plasmid DNA expressing Tumor Necrosis Factor-α (TNF-α) could enhance the effect of ionizing radiation in an aggressive, rapidly growing glioma tumor (C6) model. Plasmid DNA is an attractive vector system for delivery of foreign genes into eukaryotic cells due to its advantages including low immunogenicity in vivo, lesser toxicity and relatively low cost. We have constructed a plasmid-based mammalian expression vector (pGL1-TNF) to secrete human TNF-α from cancer cells. Plasmid DNA was successfully transfected into C6 cells in vitro by using a polyamine-facilitated method. Expression from transfected cells was detected for up to 7 days and averaged 0.4 ng TNF-a in the conditioned medium (CM) from 1 x 105 cells. Using Laser Scanning Cytometry, we confirmed that both P55 and P75 receptors for TNF-α were present on the C6 cell membrane. P55 receptors are expressed at significantly higher levels than P75 receptors. The expressed TNF-α alone did not inhibit growth of the transfected C6 cells; neither did it modulate radiation-induced inhibition of C6 cell proliferation in vitro. When plasmid pGL1-TNF was injected intratumorally, the injected tumors expressed significant amount of TNF-α. The TNF expressed was measurable up to 7 days after injection. In the in vivo tests, pGL1-TNF-α injected intratumorally 18-24 hr before radiation (Cobalt or Proton) significantly inhibited the C6 xenograft tumor progression compared to control groups. This effect was more than additive, since pGL1-TNF alone did not slow tumor growth. Blood and spleen cell population analyses showed significant change in percentages of Nature Killer and B cells in blood and spleen. Tumor histopathology revealed an earlier necrosis appearance in combination-treated tumors. The results suggest the radiation sensitizing effect of TNF-α in vivo is indirect, through multiple routes rather than any single major factor. Future studies should focus on improving the current treatment regimen and to explore the combination of radiation and other genetically delivered antitumor agents against more different forms of tumor