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).