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Iron imaging reveals tumor and metastasis macrophage hemosiderin deposits in breast cancer

Fig 3

Imaging HLM deposits in lung metastasis.

(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).

Fig 3

doi: https://doi.org/10.1371/journal.pone.0184765.g003