Targeting Leukemia-Associated Hypoxia with PR-104 Figure 4. Hypoxia promotes resistance of leukemia cell lines to chemotherapy. Cytotoxic effect of chemotherapy under normoxic and hypoxic conditions. Nalm6, REH ALL cells were treated with various chemotherapy drugs for 48 hours at 21% or 1% O2. Percentage of growth inhibition was calculated based on cell number. A and B: cell number and percentage of Annexin V–positive cells were determined by FACS. Error bars, SEM. * P,0.05; ** P,0.01; *** P,0.001. doi:10.1371/journal.pone.0023108.g004 undetectable in the tissues examined (Figure S5B) indicating that PR-104 treatment not only prolonged survival but also resulted in marked reduction of tissue infiltrating leukemia cells. Assessment of hypoxia levels in the BM by PIM staining (Fig S5C) showed that the BMs of control mice were extensively hypoxic, which contrasted with the smaller, more discrete hypoxic areas observed in the BM of treated mice. Discussion Using the hypoxia marker pimonidazole, we have shown in several in vivo models of human and murine leukemia that the BM becomes highly hypoxic in advanced stages of the leukemia process. Furthermore, BM from ALL patients stained for HIF-1a showed very strong positivity at diagnosis that was impressively reduced or eliminated when the patients achieved CR. This is the first report indicating that the expansion of hypoxic areas is one of the essential characteristics of the leukemic microenvironment. The mechanism responsible for this expansion remains elusive at this point, but one possible explanation is that accumulation of leukemic blasts in the BM leads to increased oxygen consumption, thus lowering steady-state oxygen concentrations. Time-course studies using a blast crisis CML model support this hypothesis, clearly demonstrating that evolving hypoxic areas are associated with leukemia progression. Although not thoroughly investigated in our studies, it is also likely that leukemia propagation is associated with derangements and non-functionality of vascular architecture which might contribute to progressive hypoxia despite stimulation of angiogenesis through HIF-1a. This notion is supported by findings by Schaefer et al. [36] demonstrating time-dependent alterations in the microvasculature in the in vivo leukemia models, with the early angiogenic wave followed by decreased vessel density and reduced tissue perfusion at the late stages of leukemia. We have observed that not only leukemic cells but also the surrounding stroma expresses HIF-1a (Fig. S3B), implying that hypoxia is an intrinsic property of the leukemia microenvironment. While HIF-1a expression is frequently pro- moted in normoxic tumor cells, for example through oncogenic stimuli, generating molecular signatures resembling hypoxic response in the absence of hypoxia (coined as ‘‘pseudohypoxia’’ [37–39]), hypoxic marker Pimonidazole can only be metabolized and form intracellular adducts at low oxygen concentrations, thus reflecting true hypoxia as opposed to pseudohypoxia. In a recent report, Hu et al. investigated the BM microenvironment in multiple myeloma models and observed vast hypoxic expansion (revealed by PIM staining), while na¨ıve mice did not exhibit PIM positivity [40]. In agreement with this, Colla et al showed that BM hypoxia and high HIF-1a expression is a characteristic of multiple myeloma patients [41]. Therefore, a hypoxic microenvironment may be a common phenomenon in bone marrows with expanding tumor cell populations. We also noted only infrequent HIF-1a positive cells in normal BMs supporting the notion that under PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23108