Citation: Godinho-Pereira, J.; Vaz, D.; Figueira, I.; Aniceto-Romão, J.; Krizbai, I.; Malhó, R.; Rocha, J.; Carvalheiro, M.C.; Simões, S.; Gaspar, M.M.; et al. Breast Cancer Brain Metastases: Implementation and Characterization of a Mouse Model Relying on Malignant Cells Inoculation in the Carotid Artery. Cells 2023, 12, 2076. https://doi.org/ 10.3390/cells12162076 Academic Editor: Michael Leitges Received: 31 May 2023 Revised: 17 July 2023 Accepted: 3 August 2023 Published: 16 August 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Breast Cancer Brain Metastases: Implementation and Characterization of a Mouse Model Relying on Malignant Cells Inoculation in the Carotid Artery Joana Godinho-Pereira 1,2,† , Daniela Vaz 1,2,† , Inês Figueira 1,3 , Joana Aniceto-Romão 1,2 , Istvan Krizbai 4,5 , Rui Malhó 6 , João Rocha 1,7 , Manuela Colla Carvalheiro 1,7 , Sandra Simões 1,7 , Maria Manuela Gaspar 1,7 and Maria Alexandra Brito 1,2, * 1 iMed.ULisboa—Research Institute for Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal; joanagpereira@ff.ulisboa.pt (J.G.-P.); danielaa.vaz22@gmail.com (D.V.); ines.figueira88@gmail.com (I.F.); joana.romao@live.com.pt (J.A.-R.); jrocha@ff.ulisboa.pt (J.R.); manuela.colla@campus.ul.pt (M.C.C.); ssimoes@ff.ulisboa.pt (S.S.); mgaspar@ff.ulisboa.pt (M.M.G.) 2 Department of Pharmaceutical Sciences and Medicines, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal 3 Farm-ID—Faculty of Pharmacy Research and Development Association, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal 4 Institute of Biophysics, Biological Research Centre, Eötvös Loránd Research Network (ELKH), 6726 Szeged, Hungary; krizbai.istvan@brc.hu 5 Institute of Life Sciences, Vasile Goldis Western University of Arad, Str. Liviu Rebreanu 86, 310414 Arad, Romania 6 BioISI—Biosystems and Integrative Sciences Institute, Faculty of Sciences, Universidade de Lisboa, Campo Grande, 1746-016 Lisbon, Portugal; r.malho@fc.ul.pt 7 Department of Pharmacy, Pharmacology and Health Technologies, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal * Correspondence: mariabrito@campus.ul.pt; Tel.: +351-217946449 † These authors contributed equally to this work. Abstract: Breast cancer (BC) brain metastases (BCBM) is a severe condition frequently occurring in the triple-negative subtype. The study of BCBM pathogenesis and treatment has been hampered by the difficulty in establishing a reliable animal model that faithfully recapitulates the preferential formation of brain metastases. The injection of BC cells in the carotid artery of mice has been proposed but the procedure is challenging, with the metastatic pattern being scarcely characterized. In this work, we thoroughly describe an improved procedure, highlighting the tricks and challenges of the process, and providing a characterization of the brain and peripheral metastatic pattern at the cellular and molecular level. Triple-negative BC (4T1) cells were inoculated in the common carotid artery of BALB/c mice. Brains and peripheral organs were harvested at 7–14 days for the histological characterization of the metastases’ pattern and the immunofluorescence analysis of specific markers. With our surgical procedure, both mouse death and procedure-associated weight loss were negligible. Brain metastases mostly occurred in the hippocampus, while sparse peripheral lesions were only detected in the lungs. Brain-colonizing BC cells presented proliferative (Ki-67) and epithelial (pan- cytokeratin and tomato lectin) features, which account for metastases’ establishment. The presented surgical approach constitutes an important and reliable tool for BCBM studies. Keywords: blood–brain barrier; brain metastases; breast cancer; Ki-67; intracarotid injection; mouse model; cytokeratin; peripheral metastases; proliferation; tomato lectin 1. Introduction Breast cancer (BC) is the most diagnosed and leading cancer in women, with 2.3 mil- lion new cases estimated in 2020 [1]. With the increased BC patients’ survival due to Cells 2023, 12, 2076. https://doi.org/10.3390/cells12162076 https://www.mdpi.com/journal/cells