American Association for Cancer Research
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Supplementary Methods from Nanobody-Based Targeting of the Macrophage Mannose Receptor for Effective In Vivo Imaging of Tumor-Associated Macrophages

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journal contribution
posted on 2023-03-30, 21:13 authored by Kiavash Movahedi, Steve Schoonooghe, Damya Laoui, Isabelle Houbracken, Wim Waelput, Karine Breckpot, Luc Bouwens, Tony Lahoutte, Patrick De Baetselier, Geert Raes, Nick Devoogdt, Jo A. Van Ginderachter

PDF file - 125K, This file contains Supplementary Methods, used for experiments only shown in the Supplementary Figures



Tumor-associated macrophages (TAM) are an important component of the tumor stroma and exert several tumor-promoting activities. Strongly pro-angiogenic TAMs that reside in hypoxic tumor areas highly express macrophage mannose receptor (MMR, CD206). In this study, we targeted MMR+ TAMs using nanobodies, which are single-domain antigen-binding fragments derived from Camelidae heavy-chain antibodies. MMR-specific nanobodies stained TAMs in lung and breast tumor single-cell suspensions in vitro, and intravenous injection of 99mTc-labeled anti-MMR nanobodies successfully targeted tumor in vivo. Retention of the nanobody was receptor-specific and absent in MMR-deficient mice. Importantly, co-injection of excess unlabeled, bivalent anti-MMR nanobodies reduced nanobody accumulation in extratumoral organs to background levels, without compromising tumor uptake. Within tumors, the 99mTc-labeled nanobodies specifically labeled MMR+ TAMs, as CCR2-deficient mice that contain fewer TAMs showed significantly reduced tumor uptake. Further, anti-MMR nanobodies accumulated in hypoxic regions, thus targeting pro-angiogenic MMR+ TAMs. Taken together, our findings provide preclinical proof of concept that anti-MMR nanobodies can be used to selectively target and image TAM subpopulations in vivo. Cancer Res; 72(16); 4165–77. ©2012 AACR.