Changes in tumor oxygen state after sorafenib therapy evaluated by 18F-fluoromisonidazole hypoxia imaging of renal cell carcinoma xenografts

Abstract

A mechanistic dissociation exists between tumor starvation and vascular normalization after antiangiogenic therapy. Thus, improved understanding of tumor responses (tumor starvation or vascular normalization) is important for optimizing treatment strategies. ¹⁸F-fluoromisonidazole (¹⁸F-FMISO) is widely used for imaging tumor hypoxia. To clarify the tumor response to the antiangiogenic drug sorafenib, the present study evaluated the changes in the tumor oxygen state using ¹⁸F-FMISO in mice bearing a renal cell carcinoma xenograft (A498). Mice bearing A498 xenografts were assigned to the control and three sorafenib-treatment groups and administered sorafenib (0, 10, 20 or 40 mg/kg/day, per os) once daily for 3 days. Following one day after the final administration, the mice were injected with ¹⁸F-FMISO and pimonidazole (a hypoxia marker). ¹⁸F-FMISO accumulation in the tumor was determined by autoradiography. Immunohistochemistry of pimonidazole and cluster of differentiation (CD)31 (a vascular marker) was also performed. ¹⁸F-FMISO accumulation levels in the tumor significantly increased by 4.3-, 8.4- and 8.6-fold compared with in the control group following 10, 20 and 40 mg/kg sorafenib treatments, respectively [0.07±0.04, 0.32±0.11, 0.62±0.15 and 0.63±0.23 (%ID/m²) × kg for the control, and 10, 20 and 40 mg treatments, respectively; all P<0.0083 vs. the control]. The number of pimonidazole-positive cells also significantly increased by 6.8-, 12.3- and 20.2-fold compared with in the control group following 10, 20 and 40 mg/kg sorafenib treatments, respectively (0.78±0.79, 5.36±2.29, 9.66±1.58 and 15.85±4.59% pimonidazole-positive cells; all P<0.0083 vs. the control). The number of microvessels in tumors markedly decreased to 33.5, 17.6, and 14.0% of the control following 10, 20 and 40 mg/kg sorafenib treatments, respectively (17.1±2.5, 5.7±1.0, 3.0±1.0 and 2.4±0.3 vessels/mm²; P<0.0083 vs. the control). The ¹⁸F-FMISO expression level in the tumor increased sorafenib-dose-dependently, which is consistent with the increase in the number of pimonidazole-positive cells and decrease in the number of microvessels. These findings indicated that the present sorafenib treatment protocol induces 'tumor hypoxia/starvation' in the renal cell carcinoma xenograft (A498) due to its antiangiogenic properties.

Keywords: 18F-fluoromisonidazole; antiangiogenic therapy; positron emission tomography; renal cell carcinoma; sorafenib; tumor hypoxia.

Figure 1.

Experimental protocol. RCC, renal cell carcinoma; n, number; ¹⁸F-FMISO, ¹⁸F-fluoromisonidazole; ARG, autoradiography; IHC, immunohistochemistry; H&E, hematoxylin and eosin; CD, cluster of differentiation.

Figure 2.

Representative images of ¹⁸F-FMISO ARG (upper panel), pimonidazole IHC (second panel), H&E staining (third panel) and CD31 (lower panel). The dotted and solid lines represent the tumor and muscle outlines, respectively. The black arrowheads represent the microvessels. Control, control group; Treat-10 mg, 10 mg/kg sorafenib-treated group; Treat-20 mg, 20 mg/kg sorafenib-treated group; Treat-40 mg, 40 mg/kg sorafenib-treated group. ¹⁸F-FMISO, ¹⁸F-fluoromisonidazole; H&E, hematoxylin and eosin; ARG, autoradiography; IHC, immunohistochemistry; CD, cluster of differentiation.

Figure 3.

Quantitative analysis of intratumoral (A) ¹⁸F-FMISO expression level, (B) percentage of pimonidazole-positive cells, (C) percentage of necrotic area and (D) mean vessel density (vessels/mm²). Data are expressed as the mean ± standard deviation. One-way factorial analysis of variance revealed significant changes in intratumoral ¹⁸F-FMISO expression level (A: F=28.259; P<0.0001), percentage of pimonidazole-positive cells (B: F=47.496; P<0.0001) and vessels/mm² (D: F=122.780; P<0.0001) among the four groups. No significant differences were observed in the percentage of necrotic area among the four groups (C: F=2.698; P=0.0718). *P<0.0083, by the Bonferroni post hoc test. ¹⁸F-FMISO, ¹⁸F-fluoromisonidazole; Control, control group; Treat-10 mg, 10 mg/kg sorafenib-treated group; Treat-20 mg, 20 mg/kg sorafenib-treated group; Treat-40 mg, 40 mg/kg sorafenib-treated group.