Hemosiderin iron is a biomarker of macrophage deposits in systemic and tumor microenvironments.

<p>(a) Prussian blue iron(III) and CD68 macrophage histology from orthotopic MMTV-PyMT mammary tumor, liver, and spleen. Scale bar 200μm. (b) The total number of Prussian blue iron(III)⁺ and CD68⁺ macrophages per mm² of the whole tissue cross-sectional area measured by automated cell counting. Points are individual animals (mean±s.e.m.; n = 8 animals; n.s., not significant *p<0.05, ***p<0.001 two-tailed unpaired t-test). (c) Iron(III) histochemistry beside single color channel and merged CD68, CD206, and AIF1 immunofluorescent markers. Liver (arrows denote iron(III)⁺ features) and spleen scale bar 50μm, mammary tumor scale bar 20μm. (d) The Prussian blue iron(III) images of MMTV-PyMT mammary tumors, livers, and spleens processed by down-sampling the resolution of the digitized histology to the MRI pixel scale (1:100, 0.01mm²/pixel) to generate resolution-matched clusters centered around iron(III)⁺ HLM deposits (solid lines). Scale bar 100μm. Automated size distribution analysis of (e) cross-sectional area of the clusters, and (f) frequency of the resolution-matched HLM clusters. Each point is a cluster detected in whole cross-sections of orthotopic MMTV-PyMT mammary tumors, livers, and spleens (mean±s.e.m.; n = 8 animals; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 Mann-Whitney test). (g) The number of Prussian blue iron(III)⁺ and CD68⁺ macrophages per mm² of the total cluster area. Points are individual animals (mean±s.e.m.; n = 8 animals; n.s., not significant *p<0.05 two-tailed unpaired t-test).