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Intertumoral Differences in Hypoxia Selectivity of the PET Imaging Agent ⁶⁴Cu(II)-Diacetyl-Bis(N⁴-Methylthiosemicarbazone)

¹ Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina; ² Department of Radiology, Duke University Medical Center, Durham, North Carolina; and ³ Center for In Vivo Microscopy, Duke University Medical Center, Durham, North Carolina

View larger version (109K): [in a new window]   FIGURE 1.  Image thresholding in area fraction measurement. (A and B) Original 64Cu-ATSM autoradiography image and EF5 fluorescent staining image. (C and D) Corresponding histograms for above images. The Otsu automatic thresholding method was used to determine the threshold based on histograms. Pixels with intensity higher than the threshold (in white color) were treated as positive uptake or staining area. (E and F) Segmented images using the Otsu threshold for autoradiography and EF5 staining image. Area in white is regarded as 64Cu-ATSM uptake area or positive staining area.

View larger version (28K): [in a new window]   FIGURE 2.  Biodistribution of 64Cu-ATSM in tumor-bearing rat model. (A) Dynamic uptake of 64Cu-ATSM in different organs in 9L tumor-bearing rat measured from whole-body microPET. (B) Biodistribution data measured using autoradiography on organs taken from animals at 1 h after radiopharmaceutical injection in 3 tumor lines. Biodistribution data are presented as a relative uptake value for each organ normalized to muscle in each animal.

View larger version (52K): [in a new window]   FIGURE 3.  microPET of 3 different tumor types. (A) Typical PET image for R3230Ac with transverse, coronal, and sagittal sections. An ROI was drawn in transverse section to measure the uptake level. For each tumor, measurements were taken in 4 ROIs and averaged, representing an overall uptake level. (B) Dynamic uptake of 64Cu-ATSM in 3 tumors including R3230Ac (n = 8), 9L (n = 7), and FSA (n = 7). Uptake value in muscle was taken from R3230Ac animals.

View larger version (91K): [in a new window]   FIGURE 4.  Typical immunostaining of EF5 binding in tumor tissues (R3230Ac). Dual fluorescence images (A) are overlaid to show distribution of hypoxia relative to vasculature. Hypoxic tissue is indicated by EF5 staining in orange. Vasculature is indicated by the Hoechst 33342 perfusion marker in blue. (B) A whole tumor section staining image was generated by stitching together images from microscope stage scans.

View larger version (57K): [in a new window]   FIGURE 5.  Comparisons between 64Cu-ATSM uptake and hypoxia measured by immunostaining in R3230Ac and FSA. Close correlation between 64Cu-ATSM uptake and EF5-stained hypoxic area was observed in R3230Ac tumor (left), whereas no correlation was found in FSA tumor (right). Images include 64Cu-ATSM microPET image, autoradiography (AR) section from same tumor, EF5 and Hoechst immunostaining from adjacent section, fused image from autoradiography and EF5 images, H&E staining, and correlation plot between autoradiography and EF5 staining images. EF5-stained hypoxic area is indicated by orange, perfused vessels are marked by blue fluorescent Hoechst 33342 dye, and 64Cu-ATSM distribution in AR is indicated by green in fused image. In FSA, a large amount of 64Cu-ATSM accumulated in well-perfused areas, which are indicated in Hoechst perfusion image. The spatial correlation between autoradiography and EF5 staining images in this specific FSA tumor is 0.05, whereas the spatial correlation is 0.78 in the shown R3230Ac tumor.

View larger version (103K): [in a new window]   FIGURE 6.  CA-IX and pimonidazole staining on FSA and R3230Ac tumor sections. (A and B) 64Cu-ATSM autoradiography (AR) and CA-IX staining (green) on same FSA tumor section (in same tumor as shown in Fig. 5, FSA panel). CA-IX staining shows a hypoxia distribution that is consistent with EF5 staining. (C and D) 64Cu-ATSM autoradiography and pimonidazole staining (orange) with Hoechst 33342 vascular perfusion marker (blue) illustrate low correlation between 64Cu-ATSM and this hypoxia marker in FSA. (E and F) 64Cu-ATSM autoradiography and pimonidazole staining in R3230Ac tumor. Close correlation can be seen between 64Cu-ATSM uptake and pimonidazole staining.

View larger version (56K): [in a new window]   FIGURE 7.  64Cu-ATSM distribution in response to oxygenation. FSA-bearing rats breathed carbogen for 3 h before and during microPET. (A) 64Cu-ATSM autoradiography image. (B) EF5 binding (orange) and Hoechst perfusion (blue) fluorescent staining image for the neighboring tumor section from same animal. There is no correlation between 64Cu-ATSM uptake and hypoxia marked by EF5 binding. A significant decrease in EF5 staining, but not uptake of 64Cu-ATSM, was found in carbogen-breathing animal group (Table 3).