Abstract
Hypoxia is a hallmark of many solid malignancies and confers resistance to radiotherapy as well as other treatment regimens. We investigated the combined treatment modality of ionizing radiation (IR) with the clinical stage hypoxia-activated prodrug (HAP; evofosfamide) in vitro and in vivo using mechanistic and efficacy-oriented endpoints. Based on the current insights, the combined treatment modality of IR with this HAP might lead to complementary tumor cell killing. Ionizing radiation will primarily target well-oxygenated tumor cells, while the HAP will primarily kill hypoxic tumor cells. At the same time enhanced, supra-additive cytotoxicity will be expected by the diffusible activated prodrug leading to more complex DNA damage when used in combination with IR.
METHODS
We investigated the cytotoxic effects of evofosfamide on tumor cell (A549 lung carcinoma and UT-SCC-14 head&neck squamous cell carcinoma, HNSCC) proliferation and clonogenicity in normoxic (21% O2) and hypoxic (0.2% O2) conditions. For in vivo experiments, cells (4×106) were subcutaneously injected on the back of athymic nude mice. Treatment was initiated when tumors reached volume of 300 mm3 +/- 10%. Irradiation was performed with either a fractionated (3×2Gy) or a single high dose regimen (1×10Gy). Evofosfamide was administered i.p. Q3Dx5 (50 mg/kg in saline), control mice were treated i.p. with saline.
RESULTS
Lung carcinoma A549 cells were more sensitive to evofosfamide when incubated with the compound under hypoxic conditions in comparison to normoxic conditions in a dose- and time-dependent manner. We observed decreased clonogenicity of A549 cells with evofosfamide/IR co-treatment, which suggests that the evofosfamide has a radiosensitizing effect in vitro. Moreover, evofosfamide alone and in combination with IR induced a strong DNA-damage response (γH2AX foci staining) and senescence (β-galactosidase staining) in A549 cells. We performed in vivo, efficacy- oriented experiments with lung A549 and HNSCC UT-SCC-14 xenografts to test different schedules of evofosfamide in combination with fractionated and single high-dose IR (neoadjuvant, concomitant, adjuvant). Combined treatment resulted in a strongly enhanced tumor growth delay when compared to the single treatment regimens in lung A549 xenografts applying the three different scheduling regimens in combination with fractionated and single high dose IR. Interestingly, the evofosfamide did not reduce tumor growth of HNSCC UT-SCC-14 xenografts suggesting that the response to evofosfamide and IR is highly dependent on the tumor type.
CONCLUSIONS
Enhanced tumor growth delay of lung carcinoma xenografts in response to a combined treatment of evofosfamide with two different regimens of IR suggests a potent complementary effect with IR of this compound in vivo. Further studies to investigate DNA-damage related endpoints in response to evofosfamide alone and in combination with IR in a genetically defined background (BRCA1/2 deficient cells) are ongoing to further understand the mechanism of a combined treatment. These efficacy- and mechanistic-oriented experiments on the preclinical level are highly relevant to launch clinical phase I/II trials combining radiotherapy with this clinical stage HAP.
Citation Format: Katarzyna J. Nytko-Karouzakis, Ivo Grgic, Janosch Ott, Martin Pruschy. The combined treatment modality of a hypoxia-activated prodrug (Evofosfamide) with ionizing radiation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A163.
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