Figure 3.

Hypointensities in MR images were correlated with iron-labeled CD68-positive tumor-associated macrophages (TAMs). MRI was performed on another group of mice on days 5, 7 and 14 after injection of superparamagnetic iron oxide (SPIO) nanoparticles (total n = 9, n = 3 for each time point). After scanning, animals were sacrificed to remove the tumors, which were sectioned according to where the images were acquired. (a) T₂*-weighted images (T2*WI) show hypointensities (red arrows) in tumor tissue after SPIO injection. (b) Co-staining of Prussian blue (PB) and antibody against CD68 was used to identify the location of iron particles and CD68-positive TAMs in tumor tissue. (c) Enlarged views of the regions indicated by black squares in (b), showing the co-localization of the blue iron deposits (red arrows) and the hypointensities on the images. PB-positive and CD68-negative cells were not frequently observed. (d) Enlarged views of the regions indicated by red squares in (c), showing the cell-like structure containing blue iron particles (red arrows) and CD68 positivity in the growing tumor. PB and CD68 positivity were strongly co-localized regardless of the tumor stage, although many CD68-positive cells were located in areas without a PB reaction. Dotted circle, implanted tumor fragment; black arrowhead, hemorrhage; asterisk, vessel-like structure; red arrow, CD68-positive TAMs co-localized with PB positivity; black arrow, CD68-positive TAMs not co-localized with PB positivity.