Domenici, GiacomoLopes, Nuno F.Trindade, GonçaloRamella Gal, IsabellaMiret Minard, JoanRebelo, Sofia P.Freitas, CatarinaDuarte, NádiaBrito, Catarina2025-04-102025-04-102024-122701-0198PURE: 110479819PURE UUID: 10fed2a9-fe54-4bf9-b19f-15c8fb6fdea2Scopus: 85210396978PubMed: 39601467http://hdl.handle.net/10362/182039Funding Information: G.D. and N.F.L. contributed equally to this work and share co-first authorship. The authors acknowledge funding from the Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia/ Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020), the grant PTDC/BTM-TEC/0432/2021 and the PhD scholarships 2020.07623.BD to NL, 2022.11642.BD to GT, and 2022.12962.BD to IRG. The authors also acknowledge funding from iBET (iBETXplore Starting grant; PI-3D-Abc-717). The authors thank Dr. Gabriela Silva for her critical review of the manuscript and fruitful scientific discussion. Funding Information: G.D. and N.F.L. contributed equally to this work and share co\u2010first authorship. The authors acknowledge funding from the Funda\u00E7\u00E3o para a Ci\u00EAncia e a Tecnologia/ Minist\u00E9rio da Ci\u00EAncia, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020), the grant PTDC/BTM\u2010TEC/0432/2021 and the PhD scholarships 2020.07623.BD to NL, 2022.11642.BD to GT, and 2022.12962.BD to IRG. The authors also acknowledge funding from iBET (iBETXplore Starting grant; PI\u20103D\u2010Abc\u2010717). The authors thank Dr. Gabriela Silva for her critical review of the manuscript and fruitful scientific discussion. Publisher Copyright: © 2024 The Author(s). Advanced Biology published by Wiley-VCH GmbH.Targeted, combinatorial, and immunomodulatory therapies, such as antibody-drug conjugates (ADCs) and immunomodulatory antibodies (Abs), are powerful weapons against tumor cells and immune cells within the tumor microenvironment (TME). Therefore, the evaluation of such therapies should be conducted in pre-clinical models able to recapitulate the complex cellular and molecular crosstalk of the TME. To build-in critical hallmarks of the TME, a breast cancer heterotypic 3D cell model (3D-3) is devised using a microencapsulation strategy with an inert biomaterial (alginate) and agitation-based cultures. Both stromal and immune components are added to multicellular tumor spheroids, therefore fostering cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) immunomodulatory interactions. The potential of the methodology to assess Ab-based therapies is then addressed by employing a series of anti-HER2-based ADCs. ADCs induced tumor-cell specific cytotoxicity toward HER2+ breast cancer spheroids while sparing HER2-negative CAFs. In addition, an immunomodulatory blocking Ab against colony-stimulating factor 1 receptor (CSF1R) decreases the expression of immunosuppressive and anti-inflammatory markers in TAMs, like what is previously observed upon in vivo α-CSF1R administration. Collectively, the human TME-based 3D-3 cell model is a suitable tool to evaluate the anti-tumor and immunomodulatory potential of novel antibody-based therapies directed against TME targets, such as cancer cells and macrophages.3908791eng3D cancer modelsantibody-drug conjugatescancer-associated fibroblaststargeted therapiestumor-associated macrophagesBiomaterialsBiomedical EngineeringGeneral Biochemistry,Genetics and Molecular BiologySDG 3 - Good Health and Well-beingjournal article10.1002/adbi.202400431https://www.scopus.com/pages/publications/85210396978