Effect of low dose metronomic cyclophosphamide therapy on tumor hypoxia. HIF1-α levels in tumor lysates were not significantly different between CTX and control tumors for either SW480 (A) or WM239 (B). Representative images of tumor cross sections immunostained for CA-IX (dark reaction product) and counterstained with hematoxylin: WM239 CTX (C) and control (D); SW480 CTX (E) and control (F). Insert of panel (C) is negative control without primary antibody. Scale bars = 300 μm. G) Quantification of hypoxic regions (Mean; SEM) in SW480 and WM239 xenografts from CTX and control mice. Treated WM239 xenograft tumors had significantly lower average percent hypoxic regions than control (*p < 0.05). No significant difference was observed between CA-IX staining in SW480 CTX and control tumors. H, I) Measurement of conditioned medium by ELISA demonstrates that 24 hour treatment in vitro with CoCl2 induced up-regulation in VEGF expression in both cell lines (H; SW480; I, WM239), while treatment with low dose 4-HC does not. Combining these treatments had no additive effect over the induction produced by CoCl2 alone.