Targeting mannose receptor expression on macrophages in atherosclerotic plaques of apolipoprotein E-knockout mice using 111In-tilmanocept

Abstract

Background: Atherosclerotic plaque phenotypes are classified based on the extent of macrophage infiltration into the lesions, and the presence of certain macrophage subsets might be a sign for plaque vulnerability. The mannose receptor (MR) is over-expressed in activated macrophages. Tilmanocept is a tracer that targets MR and is approved in Europe and the USA for the detection of sentinel lymph nodes. In this study, our aim was to evaluate the potential of ¹¹¹In-labelled tilmanocept for the detection of MR-positive macrophages in atherosclerotic plaques of apolipoprotein E-knockout (ApoE-KO) mouse model.

Methods: Tilmanocept was labelled with ¹¹¹In. The labelling stability and biodistribution of the tracer was first evaluated in control mice (n = 10) 1 h post injection (p.i.). For in vivo imaging studies, ¹¹¹In-tilmanocept was injected into ApoE-KO (n = 8) and control (n = 8) mice intravenously (i.v.). The mice were scanned 90 min p.i. using a dedicated animal SPECT/CT. For testing the specificity of ¹¹¹In-tilmanocept uptake in plaques, a group of ApoE-KO mice was co-injected with excess amount of non-labelled tilmanocept. For ex vivo imaging studies, the whole aortas (n = 9 from ApoE-KO and n = 4 from control mice) were harvested free from adventitial tissue for Sudan IV staining and autoradiography. Cryosections were prepared for immunohistochemistry (IHC).

Results: ¹¹¹In radiolabelling of tilmanocept provided a yield of greater than 99%. After i.v. injection, ¹¹¹In-tilmanocept accumulated in vivo in MR-expressing organs (i.e. liver and spleen) and showed only low residual blood signal 1 h p.i. MR-binding specificity in receptor-positive organs was demonstrated by a 1.5- to 3-fold reduced uptake of ¹¹¹In-tilmanocept after co-injection of a blocking dose of non-labelled tilmanocept. Focal signal was detected in atherosclerotic plaques of ApoE-KO mice, whereas no signal was detected in the aortas of control mice. ¹¹¹In-tilmanocept uptake was detected in atherosclerotic plaques on autoradiography correlating well with Sudan IV-positive areas and associating with subendothelial accumulations of MR-positive macrophages as demonstrated by IHC.

Conclusions: After i.v. injection, ¹¹¹In-tilmanocept accumulated in MR-expressing organs and was associated with only low residual blood signal. In addition, ¹¹¹In-tilmanocept uptake was detected in atherosclerotic plaques of mice containing MR-expressing macrophages suggesting that tilmanocept represents a promising tracer for the non-invasive detection of macrophages in atherosclerotic plaques.

Keywords: Atherosclerosis; Inflammation; M2 differentiated macrophages; Non-invasive imaging; SPECT/CT; Tilmanocept.

Fig. 1

Chemical structure of tilmanocept. It is composed of a dextran backbone (black) and multiple units of d-mannose (blue) and DTPA (red) attached to the backbone. The d-mannose units are the receptor recognizing moieties, and the DTPA units are used for ¹¹¹In labelling

Fig. 2

SDS-PAGE analysis of ¹¹¹In-tilmanocept. 1 ¹¹¹In-tilmanocept sample, which was incubated in murine serum at 37 °C for 90 min. 2 ¹¹¹In-acetate, which was used as a low molecular weight radioactivity marker on the same gel

Fig. 3

Sagittal and coronal views of SPECT/CT images acquired in vivo 90 min after i.v. injection of ¹¹¹In-tilmanocept from ApoE-KO non-blocked, blocked and control mice. Note the intense focal signals in low abdominal atherosclerotic plaques of ApoE-KO non-blocked mice (white arrows). In contrast, no focal uptake was detected in the aortas of ApoE-KO mice after blocking with excess amount (10 nmol) of non-labelled tilmanocept and in those of control mice. Kidneys (arrowheads) liver (asterisk), spleen (square)

Fig. 4

a Sudan IV staining and corresponding autoradiography (AR) of the aortas extracted from ApoE-KO non-blocked, blocked and control mice. Whole aorta surface area was quantified using ImageJ software. b Quantification of the autoradiography signals expressed as intensity per unit area of the whole aorta (whole aorta autoradiographic signal/whole aorta area, QL/mm²)

Fig. 5

Representative images of HE, M3/84 (macrophages) and MR (CD206) immunohistochemistry. In control mice, no plaques with macrophages were observed, while fibrous/fibroatheromatous plaques were present in the aortas extracted from ApoE-KO mice. The lesions (fatty streaks and fibrous plaques) showed high amounts of MR⁺ macrophages (100 μm (bars), vascular lumen (L), intima (arrow), media (asterisk), adventitia (arrowhead))