Hemosiderin-Laden Macrophages in Canine Mammary Carcinomas

Abstract

Macrophages are among the main actors in cancer immunoediting, with several functions, including recycling iron and packaging it in hemosiderin. Even though TAMs are widely studied in breast cancer and canine mammary tumors, hemosiderin-laden macrophages (HLMs) have not received as much attention. Considering the growing interest in iron metabolism in cancer, this study aims to evaluate the presence of HLMs in canine mammary tumors. Fifty cases of canine mammary carcinomas presenting aggregates of pigmented macrophages were chosen. Prussian blue and Meguro staining were performed to assess the presence of iron. Immunohistochemistry was carried out to try to identify macrophagic phenotypes and hypothesize their role. Evaluation of the H&E sections showed that pigmented macrophages were variously localized in peritumoral and stromal areas. These pigmented cells were variably stained with Prussian blue and reacted strongly with DAB in the Meguro staining method, thus confirming the presence of iron within them. In their immunohistochemistry, the HLMs were negative for the MAC387 but positive for CD 204 and VEGF. Considering their positivity for CD 204, HLMs could be M2 macrophages that supply iron to both the neoplastic cells and the tumor inflammatory microenvironment, promoting angiogenesis and protecting cancer cells from hypoxia.

Keywords: CD204; HLMs; VEGF; dog; immunohistochemistry; iron; mammary gland; neoplasm.

Figure 1

(a) Brown-pigmented macrophages (asterisk) in a peritumoral area of grade I tubular carcinoma (H&E, 20×); (b) pigmented macrophages (asterisk) in the marginal area of stromal TILs (arrow) in a grade II tubulopapillary carcinoma (H&E, 20×); (c) pigmented macrophages (asterisk) in marginal area of a TLS with HEVs (arrowhead) in a grade III solid carcinoma (H&E, 10×); (d) details of figure (c) with pigmented macrophages (asterisk) and HEV (arrowhead) in a TLS (H&E, 40×).

Figure 2

(a) Blue staining with Prussian blue in pigmented macrophages in canine mammary carcinoma (Prussian blue, 40×) and positive control (inset, Prussian blue, 60×); (b) pigmented macrophages exhibiting brown color with Meguro stain (Meguro stain, 40×) and positive control (inset, Meguro stain, 60×); (c) purple color in pigmented macrophages with modified Meguro stain (modified Meguro stain, 40×) and positive control (inset, modified Meguro stain, 60×).

Figure 3

(a) Macrophage marker MAC387 highlighted scattered macrophages (arrowhead), but HLMs (arrow) were negative for the same antibody (DAB, 10×); (b) details of figure (a) (DAB, 40×); (c) HLMs were strongly positive for CD204 (DAB, 20×).

Figure 4

(a) HLM positivity for VEGF (DAB, 40×); (b) negative control (DAB, 40×); (c) VEGF expressed in HLMs (arrow), mast cells (arrowhead), and neoplastic epithelium (asterisk) (DAB, 20×).

Figure 5

(a) HLMs were positive for VEGFR, especially near TILs or TLS (DAB, 40×); (b) mast cell tryptase was detected in mast cells (arrowhead) and in some cytoplasmatic granules in HLMs (arrow) (DAB, 10×); (c) TGF-α positivity in numerous HLMs and endothelial cells (DAB, 20×); (d) p63 positivity in HLMs and in nuclei of myoepithelial cells (DAB, 20×).