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

Abstract

Iron-deposition is a metabolic biomarker of macrophages in both normal and pathological situations, but the presence of iron in tumor and metastasis-associated macrophages is not known. Here we mapped and quantified hemosiderin-laden macrophage (HLM) deposits in murine models of metastatic breast cancer using iron and macrophage histology, and in vivo MRI. Iron MRI detected high-iron pixel clusters in mammary tumors, lung metastasis, and brain metastasis as well as liver and spleen tissue known to contain the HLMs. Iron histology showed these regions to contain clustered macrophages identified by their common iron status and tissue-intrinsic association with other phenotypic macrophage markers. The in vivo MRI and ex vivo histological images were further processed to determine the frequencies and sizes of the iron deposits, and measure the number of HLMs in each deposit to estimate the in vivo MRI sensitivity for these cells. Hemosiderin accumulation is a macrophage biomarker and intrinsic contrast source for cellular MRI associated with the innate function of macrophages in iron metabolism systemically, and in metastatic cancer.

Fig 1. Quantitative contrast agent-free MRI of macrophage iron deposits.

(a) Multi-gradient echo (MGE) MRI image (first-echo) showing variation of contrast with increasing concentration of iron(III) (0-0-0.25 mg g^-1). (b) Quantitative MRI measurements of signal relaxation rates R₂* (= 1/ T₂*) for the MGE image series as a function of iron(III) concentration (solid-line linear fit, dashed lines 95% confidence interval). (c) Parametric iron(III) maps generated for the standards, and H₂O (dashed circle). (d) Iron MRI maps of whole blood, transferrin (Tfn, ~20μg iron/mL), macrophage hemosiderin from mouse spleen, superparamagnetic iron oxide (SPIO) nanoparticles in aqueous solution (200μg/mL), and in vivo in mammary tumors, livers (no tumor), and spleens (no tumor) of the MMTV-PyMT breast cancer models. Upper scale bar, 1mm, lower scale bar 2mm. Expansions show high-iron pixel cluster regions indicative of hemosiderin deposition. Scale bar 200μm. High-iron(III) MRI clusters quantified automatically by (e) counting the total number of high iron pixel clusters in the iron MRI images (0.15–0.3 mg iron(III) g^-1 range), and (f) measuring the average cross-sectional areas of the clusters (mm²). Points are individual animals (mean±s.e.m.; n = 8 animals; n.s. not significant p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 two-tailed unpaired t-test).

Fig 2. Hemosiderin iron is a biomarker of macrophage deposits in systemic and tumor microenvironments.

(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).

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 4. Imaging HLM deposits in brain metastasis.

(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).