Evaluation of hypoxia in an experimental rat tumour model by [(18)F]fluoromisonidazole PET and immunohistochemistry

Abstract

This study aimed to evaluate tumour hypoxia by comparing [(18)F]Fluoromisonidazole uptake measured using positron emission tomography ([(18)F]FMISO-PET) with immunohistochemical (IHC) staining techniques. Syngeneic rhabdomyosarcoma (R1) tumour pieces were transplanted subcutaneously in the flanks of WAG/Rij rats. Tumours were analysed at volumes between 0.9 and 7.3 cm(3). Hypoxic volumes were defined using a 3D region of interest on 2 h postinjection [(18)F]FMISO-PET images, applying different thresholds (1.2-3.0). Monoclonal antibodies to pimonidazole (PIMO) and carbonic anhydrase IX (CA IX), exogenous and endogenous markers of hypoxia, respectively, were used for IHC staining. Marker-positive fractions were microscopically measured for each tumour, and hypoxic volumes were calculated. A heterogeneous distribution of hypoxia was observed both with histology and [(18)F]FMISO autoradiography. A statistically significant correlation (P<0.05) was obtained between the hypoxic volumes defined with [(18)F]FMISO-PET and the volumes derived from the PIMO-stained tumour sections (r=0.9066; P=0.0001), regardless of the selected threshold between 1.4 and 2.2. A similar observation was made with the CA IX staining (r=0.8636; P=0.0006). The relationship found between [(18)F]FMISO-PET and PIMO- and additionally CA IX-derived hypoxic volumes in rat rhabdomyosarcomas indicates the value of the noninvasive imaging method to measure hypoxia in whole tumours.

Figure 1

Pimonidazole staining photographs (made with Carl Zeiss KS100 Software). (A) Peripheral view. (B) Central view. Both slices are shown on a magnification × 25. Scale bar is 40 μm. Abbreviations: N=necrosis, V=viable, well-oxygenated tumour tissue, P=PIMO-positive staining and the arrow indicates a blood vessel.

Figure 2

[¹⁸F]FMISO-PET mean tissue to heart activity ratios of the lung (n=12) , muscle (n=24; that is, front leg muscle n=12 and hind leg muscle n=12) □ and a body area 15 mm above the heart (n=12) ▪ on 12 randomly chosen 2 h p.i. images. Similarly, tumours (n=48) were analysed on 2 h p.i. images. For all the tissues, cumulative histogram analysis was carried out.

Figure 3

(A) [¹⁸F]FMISO volume defined on 2 h p.i. images, using a threshold 1.4 plotted against PIMO-positive volume (n=11 tumours). (B) r- and P-values for a range of thresholds between 1.4 and 3.0 for the same comparison. (C) [¹⁸F]FMISO volume defined on 2 h p.i. images, using a threshold 1.4 plotted against CA IX-positive volume (n=11 tumours). (D) r- and P-values for a range of thresholds between 1.4 and 3.0 for the same comparison.

Figure 4

Autoradiography images of a large (A: 4.36 cm³) and a small (B: 0.92 cm³) tumour. From the top to bottom of the images, peripheral to central images are shown. Each image is scaled to the hottest pixel (=most [¹⁸F]FMISO uptake) in the tumour. Images are enlarged to 200%.