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

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.

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.

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.

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.

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.