Hypoxia-mediated apoptosis from angiogenesis inhibition underlies tumor control by recombinant interleukin 12

The role of angiogenesis inhibition in the antitumor activity of recombinan t murine interleukin 12 (rmIL-12) was studied in K1735 murine melanomas, th e growth of which is rapidly and markedly suppressed by rmIL-12 treatment. On the basis of the prediction that tumor ischemia should result from thera peutic angiogenesis inhibition, tumor cell hypoxia was evaluated as a marke r of ischemia using the EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3-pentaf luoropropyl)acetamide] approach. This method measures intracellular binding of the nitroimidazole EF5, which covalently binds to cellular macromolecul es selectively under hypoxic conditions. Whereas 1 week of rmIL-12 treatmen t effectively inhibited K1735 cell-induced angiogenesis in Matrigel neovasc ularization assays, 2 weeks of treatment were needed before severe tumor ce ll hypoxia was detected in K1735 tumors. The hypoxia that developed was reg ional and localized to tumor areas distant from blood vessels. The great ma jority of severely hypoxic tumor cells were apoptotic, and in vitro studies indicated that the degree of hypoxia present within treated tumors was suf ficient to trigger K1735 apoptosis, Tumor cell apoptosis was also prevalent in the first week of rmIL-12 treatment when few cells were hypoxic. In vit ro studies indicated that this non-hypoxia-related apoptosis was induced di rectly by IFN-gamma produced in response to rmIL-12 administration. These s tudies reveal that rmIL-12 controls R1735 tumors initially by IFN-gamma-ind uced apoptosis and later by hypoxia-induced apoptosis, They also establish hypoxia as an expected result of tumor angiogenesis inhibition and a mediat or of its therapeutic effect.