Comparison of the Hypoxia PET Tracer (18)F-EF5 to Immunohistochemical Marker EF5 in 3 Different Human Tumor Xenograft Models

Abstract

The availability of (18)F-labeled and unlabeled 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide (EF5) allows for a comparative assessment of tumor hypoxia by PET and immunohistochemistry; however, the combined use of these 2 approaches has not been fully assessed in vivo. The aim of this study was to evaluate (18)F-EF5 tumor uptake versus EF5 binding and hypoxia as determined from immunohistochemistry at both macroscopic and microregional levels.

Methods: Three tumor models-PC3, HCT116, and H460-were evaluated. Tumor-bearing animals were coinjected with (18)F-EF5 and EF5 (30 mg/kg), and PET imaging was performed at 2.5 h after injection. After PET imaging and 2 min after Hoechst 33342 injection, the tumors were excised and evaluated for (18)F-EF5 distribution by autoradiography and EF5 binding by immunohistochemistry. Additionally, the effects of nonradioactive EF5 (30 mg/kg) on the hypoxia-imaging characteristics of (18)F-EF5 were evaluated by comparing the PET data for H460 tumors with those from animals injected with (18)F-EF5 alone.

Results: The uptake of (18)F-EF5 in hypoxic tumor regions and the spatial relationship between (18)F-EF5 uptake and EF5 binding varied among tumors. H460 tumors showed higher tumor-to-muscle contrast in PET imaging; however, the distribution and uptake of the tracer was less specific for hypoxia in H460 than in HCT116 and PC3 tumors. Correlation analyses revealed that the highest spatial correlation between (18)F-EF5 uptake and EF5 binding was in PC3 tumors (r = 0.73 ± 0.02) followed by HCT116 (r = 0.60 ± 0.06) and H460 (r = 0.53 ± 0.10). Uptake and binding of (18)F-EF5 and EF5 correlated negatively with Hoechst 33342 perfusion marker distribution in the 3 tumor models. Image contrast and heterogeneous uptake of (18)F-EF5 in H460 tumors was significantly higher when the radiotracer was used alone versus in combination with unlabeled EF5 (tumor-to-muscle ratio of 2.51 ± 0.33 vs. 1.71 ± 0.17, P < 0.001).

Conclusion: The uptake and hypoxia selectivity of (18)F-EF5 varied among tumor models when animals also received nonradioactive EF5. Combined use of radioactive and nonradioactive EF5 for independent assessment of tumor hypoxia by PET and immunohistochemistry methods is promising; however, the EF5 drug concentrations that are required for immunohistochemistry assays may affect the uptake of (18)F-EF5 in hypoxic cells in certain tumor types as observed in H460 in this study.

Keywords: 18F-EF5; EF5; PET; hypoxia; tumor.

FIGURE 1

¹⁸F-EF5 PET images of PC3 (A), HCT116 (B) and H460 (C) tumor xenografts at 2.5 h after radiotracer injection. Images are scaled to same maximal standardized uptake value (SUV), and mid-coronal sections are shown for each of 3 tumors (indicated by arrows).

FIGURE 2

Comparison of ¹⁸F-EF5 uptake with EF5 binding and hypoxia in PC3 tumor model. (A) Composite fluorescence image showing EF5 binding in green and in vivo perfusion marked by Hoechst 33342 in blue. Scale bar = 2 mm. (B) High magnification of area shown in box on image A. Arrowhead indicates blood vessel. Scale bar = 0.5 mm. (C) Autoradiography image showing ¹⁸F-EF5 uptake distribution in tumor section adjacent to that shown in A. (D) Spatial correlation analysis showing close correlation between EF5 binding and ¹⁸F-EF5 uptake in tumor sections A and C (r = 0.74). Each point in scatterplot represents 126-μm pixel and corresponding marker values on coregistered image (immunohistochemistry and DAR).

FIGURE 3

¹⁸F-EF5 uptake and hypoxia in HCT116 tumor. (A) Composite fluorescence images of EF5 and Hoechst 33342 for representative tumor section from animal shown in Figure 1B. Scale bar = 2 mm. (B) High magnification of area shown in box in image A. Scale bar = 0.5 mm. (C) Autoradiography image showing intratumoral distribution of ¹⁸F-EF5 uptake. (D) Scatterplot showing spatial relationship between the 2 hypoxia markers on adjacent tumor sections shown in A and C.

FIGURE 4

Uptake and spatial distribution of ¹⁸F-EF5 compared with EF5 binding in H460 tumor. Representative immunofluorescence (A) and autoradiography (C) images showing EF5 binding (green), vascular per-fusion (blue) and ¹⁸F-EF5 distribution (C) in tumor xenograft shown in Figure 1C. Tumor area in box on image A is shown at high magnification in B. Spatial relationship between EF5 binding and ¹⁸F-EF5 distribution determined from successive tumor sections in images A and C is shown in D. Scale bar in A = 2 mm and in B = 0.5 mm.

FIGURE 5

Correlation of ¹⁸F-EF5 uptake–positive area fraction with EF5 binding area fraction determined from adjacent tumor sections from same animal for each of 3 tumors.

FIGURE 6

Comparison of PET data for H460 tumor xenografts injected with ¹⁸F-EF5 alone or mixture of ¹⁸F-EF5 and EF5 (n = 6 animals per group). Line in box shows median value for group. *Fraction of voxels with T/M ratio > 1.5.