It has been postulated that anti-angiogenic therapies might be less susceptible to resistance compared to conventional treatment strategies, which directly target genetically unstable tumor cells. However, intrinsic and acquired resistance is commonly seen preclinically and in patients undergoing anti-angiogenic therapies. Besides the use of targeted anti-angiogenic agents, the continuous or frequent administration of comparatively low doses of conventional cytotoxic agents with no extended breaks (i.e. low-dose metronomic chemotherapy (LDM)) represents another promising anti-angiogenic approach that is being explored in clinical trials. To study the basis of resistance to LDM cyclophosphamide (CTX), the clinically most advanced agent in this setting, we derived several variants of the human prostate cancer cell line PC-3 having acquired resistance to LDM CTX following prolonged therapy of xenograft bearing mice. The resistance phenotype was stable over numerous in vitro and in vivo passages. LDM CTX induced reduction of microvessel density was similar in resistant xenografts compared to tumors of parental PC-3 and control variants (obtained by serial in vivo passage without LDM CTX therapy). RNA preparations of resistant and corresponding control tumors were subjected to microarray analysis using 19K human cDNA arrays. Genes found consistently upregulated in the LDM CTX resistant variant LCR1.1 are involved in processes such as ischemia tolerance (janus kinase 1, calmodulin 2), transmembrane glucose transport (glut-12),glycolysis (enolase superfamily member 1) and cell survival upon growth factor deprivation (apoptosis inhibitor 5). Increased expression of downstream components of the mTOR signaling pathway (members of the eukaryotic translation initiation factor family) suggests a potential role of this pathway in LDM CTX resistance. These expression changes support the notion that reduced vascular dependence, an adaptive tumor cell response to decreased oxygen, nutrient and growth factor availability resulting from anti-angiogenic therapy, might be implicated in resistance to such therapies, in particular metronomic protocols. If confirmed in ongoing clinical trials, our results could have major implications for the selection of patients most suitable for anti-angiogenic therapies and the management of emerging resistance.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA