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) of brain metastasis from MMTV-PyMT mammary-brain model (intracardiac injection). Scale bar 1 mm. Expansion shows individual metastasis, and individual iron(III)+ HLM deposit (5x). Scale bar 250μm. (b) The total number of iron(III)+ HLMs and CD68+ macrophages per mm2 brain metastasis measured by automated cell counting. Points are individual metastases. (mean±s.e.m.; n = 86 iron(III)+ and CD68+ metastasis; n = 16 brain sections, n = 5 animals; ****p<0.0001 Mann-Whitney test). (c) Iron(III) histochemistry beside single color channel and merged CD68, CD206, and AIF1 immunofluorescent markers. Scale bar 50μm. (d) In vivo iron(III) MRI maps measured in MMTV-PyMT mammary-brain metastasis models. Scale bar 2mm. Expansion shows high-iron(III) cluster regions and individual metastases. (e) High-iron(III) MRI clusters automatically counted in normal brain regions and the metastases, and (f) the average cross-sectional areas of the high-iron(III) clusters (mean±s.e.m.; n = 97 metastasis, n = 26 high-iron(III) clusters; n = 66 normal regions, n = 6 high-iron(III) clusters, n = 19 animals; **p<0.001 Mann-Whitney test). (g) Whole brain cross sections of the metastasis models stained for Prussian blue iron(III). The processed images show individual metastasis (outlines), HLM deposits (arrows), and MRI 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 (h) the average HLM cluster cross-sectional area measured per brain metastasis, and (i) number of iron(III)+ HLMs per cluster (mean±s.e.m.; n = 46 brain metastasis; n = 212 HLM clusters, n = 16 brain sections, n = 5 animals).