Axitinib sensitization of high Single Dose Radiotherapy

Abstract

Background and purpose: Single Dose Radiation Therapy (SDRT) provides remarkably high rates of control even for tumors resistant to fractionated radiotherapy. SDRT tumor control depends on acute acid sphingomyelinase-mediated endothelial cell injury and monoclonal antibodies targeting Vascular Endothelial Cell Growth Factor (VEGF) signaling radiosensitized tumor endothelium when delivered immediately prior to irradiation. Here we evaluate the ability of the oral VEGF receptor inhibitor, axitinib, to sensitize tumor endothelium and increase tumor control with SDRT.

Methods and materials: Axitinib was added to primary cultured endothelial cells, or administered orally to Sv129/BL6 mice bearing radiosensitive MCA/129 sarcoma or radioresistant B16F1 melanoma flank tumors, followed by SDRT. Endothelial apoptosis was assessed by TUNEL assay or bis-benzamide staining. Mice with irradiated tumors were followed for 90days to evaluate the impact of axitinib on SDRT tumor control.

Results: Pre-treatment with axitinib increased acute endothelial cell apoptosis following SDRT in vitro, and in vivo for both MCA/129 and B16F1 tumors. Axitinib correspondingly increased SDRT tumor growth delay and complete response rate (by 40%) for both tumors. Administration precisely 1h before SDRT was critical for radiosensitization.

Conclusions: Axitinib radiosensitizes tumor endothelial cells and enhances tumor cure with SDRT, which may permit dose de-escalation and significantly expand the range of clinical indications for SDRT.

Keywords: Acid sphingomyelinase; Anti-angiogenic; Ceramide; Radiosurgery; Stereotactic body radiation therapy; Vascular endothelial growth factor.

Fig. 1

Axitinib increases endothelial cell apoptosis after SDRT in vivo. Axitinib was administered to Sv129/BL6 mice bearing MCA/129 sarcoma or B16F1 melanoma flank tumors (100–150 mm³) and after 1 h tumors were treated with SDRT. Tumors were double-stained using TUNEL labeling and MECA-32 immunohistochemistry to identify apoptotic endothelial cells 6 h after SDRT. (A) Representative 5-µm sections of MCA/129 tumors untreated or at 6 h after 27 Gy SDRT plus axitinib. Apoptotic endothelium exhibits a brown TUNEL-positive nuclear signal surrounded by a dark blue plasma membrane signal for MECA-32 (indicated by arrows, magnification 400×). (B) Axitinib given to mice bearing MCA/129 tumors at 1 h preceding 27 Gy dose-dependently increases SDRT-induced endothelial cell apoptosis. (C) Axitinib administered 1 h preceding 40 Gy to mice bearing B16F1 tumors increases endothelial cell apoptosis. Data (mean ± SEM) are collated from 2 to 3 mice per dose with 1000–2000 endothelial cells evaluated. *p < 0.001 compared to 27 Gy alone. **p < 0.001 compared to 40 Gy alone.

Fig. 2

Axitinib pre-treatment increases complete response rate of MCA/129 sarcomas to SDRT. Sv129/BL6 mice bearing MCA/129 flank tumors (100–150 mm³) were given a single PO dose of axitinib (300 mg/kg) 1 h prior to tumor treatment with 27 Gy. Tumor volumes were measured for 90 days. Arrows indicate day of treatment. Lines represent individual tumor responses measured daily for each tumor with complete responses shown in black.

Fig. 3

Axitinib pre-treatment enables complete responses in radioresistant B16F1 melanomas after SDRT. Sv129/BL6 mice with B16F1 flank tumors were given axitinib 1 h prior to 40 Gy SDRT to the tumor. Arrows denote day of treatment. Lines represent individual tumor responses for each mouse with complete responses in black.

Fig. 4

Time dependence of axitinib administration with SDRT. Sv129/BL6 mice bearing MCA/129 flank sarcomas were treated with axitinib at various time intervals before or after 27 Gy SDRT. (A) At 6 h after SDRT, tumor endothelial cells were examined for apoptosis. Data (mean ± SEM) are collated from 2 to 4 mice per dose with 1000–2000 endothelial cells evaluated. *p < 0.001. (B) Mice were given axitinib either 1 or 2 h prior to 27 Gy SDRT (black arrow) and tumor size was measured for 60 days. Graph depicts Kaplan–Meier survival analysis of tumor progression (defined as 25% growth over baseline).