The promising treatment combination of ionizing radiation (IR) with a hypoxia-activated prodrug (HAP) is based on biological cooperation. treatment response in assessment to the related neoadjuvant and adjuvant regimens. Adjuvant evofosfamide was more potent than concomitant and neoadjuvant evofosfamide when combined with a solitary high dose of IR. Hypoxic UT-SCC-14 cells and tumor xenografts thereof were resistant to evofosfamide only and in combination with IR, most probably due to reduced P450 oxidoreductase appearance, which might take action as major predictive determinant of level of sensitivity to HAPs. In summary, evofosfamide with IR is definitely a potent combined treatment modality against hypoxic tumors. However, the effectiveness and the restorative end result of this combined treatment modality is definitely, as indicated here in preclinical tumor models, dependent on arranging guidelines and tumor type, which is definitely most probably related to the IGSF8 status of respective HAP-activating oxidoreductases. Further biomarker development is definitely necessary for the release of successful medical tests. with defined hypoxic conditions (0.2% O2). Curiously, A549 cells were also more sensitive than UT-SCC-14 to increasing concentrations of evofosfamide (Supplementary Number 2). The cytochrome P450 oxidoreductase (POR) offers previously been recognized as major determinant for the level of sensitivity of hypoxia-activated prodrugs [18, 19]. Consequently, the appearance level of POR was identified on the cellular and tumor level by western blotting and immunohistochemistry, respectively. The POR expression level was strongly reduced in UT-SCC-14 cells and UT-SCC-14-derived tumors in comparison to A549 cells and tumors derived thereof (Figure 2A, 2B). This is most probably the cause for evofosfamide-resistance against the head&neck tumor model used in this study. Furthermore, transient downregulation of POR in A549 cells with POR-directed siRNA resulted in reduced sensitivity to evofosfamide in these cells relative to control siLUC-transfected A549 cells (Supplementary Figure 3), reinforcing the role of POR for evofosfamide sensitivity. Despite several attempts, we could not perform the opposite experimental approach to overexpress POR in UTSCC-14 cells. These cells did always undergo cell death upon genetic manipulation alone. Figure 2 Differential POR- and PLGF-levels in A549 and UT-SCC-14 tumors To further analyze the differential treatment response in between A549 and UT-SCC-14-derived tumors, comprehensive analysis of hypoxia-related secreted factors was performed by Bio-plex analysis. Unfortunately, the levels of serum secreted factors in mice carrying tumor xenografts were below detection limits. Therefore, analysis of conditioned media derived from A549 and UT-SCC-14 cells was performed. The basal secretory levels of most factors analyzed were different in between the buy 3371-27-5 two cell lines (e.g. VEGF, IL-6, Osteopontin, sEGFR, TNF) and did not change in response to evofosfamide treatment (Supplementary Figure 4). Interestingly, placental growth factor (PlGF) was strongly increased in A549 but not in UT-SCC-14 cells in response to evofosfamide, suggesting that an increase of PlGF might be used as an early response biomarker (Figure ?(Figure2C2C). Next, the potency of evofosfamide was investigated in the evofosfamide-sensitive A549-derived tumor model as part of a combined treatment modality (neoadjuvant, concomitant, adjuvant) with a single high dose of IR (10 Gy). The adjuvant combined treatment modality was most effective and induced a strong tumor growth delay in comparison to evofosfamide and IR alone (experiments with A549 cells demonstrated a dose- and hypoxia incubation time-dependent antiproliferative effect of evofosfamide (Figure ?(Figure4A).4A). To determine cancer cell clonogenicity, A549 cells were incubated with evofosfamide (0.5 M) for buy 3371-27-5 4 hours under hypoxia (0.2% O2) and normoxia, respectively, followed by irradiation under reoxygenated conditions. Combined treatment of A549 cells with evofosfamide and increasing doses of IR resulted in a strong, supra-additive reduction of clonogenicity when cells were preincubated with evofosfamide under hypoxic conditions in comparison to preincubation under normoxic conditions (DEF0.1=1.44+/?0.07 vs DEF0.1 of 1.16 buy 3371-27-5 +/? 0.07 respectively, and DEF0.37=1.72+/?0.12 vs DEF0.37=1.23+/?0.24, respectively) (Figure ?(Figure4B4B). Figure 4 Treatment response to evofosfamide and irradiation (see above Figure 4B, 4C). Similar results were obtained when DNA damage was probed on the level of residual 53BP1-foci (Supplementary Figure 5). Figure 5 DNA damage in response to evofosfamide and irradiation Senescence is a well-known mode of cell death induced upon treatment with alkylating agents . A high percentage of -galactosidase positive A549 cells was induced on treatment with evofosfamide under hypoxic conditions, which was further increased on combined treatment with IR (Figure ?(Figure5B).5B). These results demonstrate that evofosfamide alone induces a strong DNA damage response and senescence in lung carcinoma cells. The small increase in the quantity of senescent cells in response to evofosfamide in mixture with IR corresponds in component with reduced clonogenicity of A549 cells in.