Iron imaging reveals tumor and metastasis macrophage hemosiderin deposits in breast cancer
(a, left) Prussian blue iron(III) histology, and (right) immunofluorescence staining (DAPI, CD68, CD31, CD3) of lung metastasis from MMTV-PyMT mammary-lung model (intravenous injection). Scale bar 100μm. Expansion shows HLM deposits. Scale bar 40μm. (b) The total number of iron(III)+ HLMs and CD68+ macrophages per mm2 lung metastasis measured by automated cell counting. Points are individual metastases. (mean±s.e.m.; n = 42 metastases, n = 8 lung sections; ****p<0.0001 Mann-Whitney test). (c) Iron(III) histochemistry beside single color channel and merged CD68, CD206, and AIF1 immunofluorescent markers. Scale bar 20μm. (d) In vivo iron(III) MRI maps measured in MMTV-PyMT mammary-lung metastasis models. Scale bare 2mm. Expansion shows high-iron(III) cluster regions and individual metastases. Scale bar 200μm. (e) High-iron(III) MRI clusters automatically analyzed to determine the average cross-sectional area of the clusters (mm2). Points are individual high-iron(III) clusters measured in metastasis and normal lung regions (mean±s.e.m.; n = 166 metastases, n = 47 normal lung regions, n = 17 mice; ****p<0.0001 Mann-Whitney test). (f) Whole lung cross sections of the metastasis models stained for Prussian blue iron(III), and processed images showing individual metastasis (outlines), HLM deposits (arrows), and resolution-matched HLM clusters in the individual metastasis (filled regions). Scale bar 1mm. Expansion scale bar 400μm. Prussian blue iron(III) histology automatically assessed for (g) metastasis cross-sectional area (mean±s.e.m. n = 169 metastases, n = 8 lung sections), (h) MRI-resolution matched HLM cluster area, and (i) number of iron(III)+ HLMs per cluster (mean±s.e.m.; n = 42 iron(III)+ metastases out of 169 counted, n = 8 lung sections).