Ncube, Keith NtokozoJurgens, TamarinSteenkamp, VanessaCromarty, Allan DuncanVan den Bout, Jan Iman2024-03-152024-03-152023-05-19Ncube, K.N.; Jurgens, T.; Steenkamp, V.; Cromarty, A.D.; van den Bout, I.; Cordier, W. Comparative Evaluation of the Cytotoxicity of Doxorubicin in BT-20 Triple-Negative Breast Carcinoma Monolayer and Spheroid Cultures. Biomedicines 2023, 11, 1484. https://DOI.org/10.3390/biomedicines11051484.2227-905910.3390/biomedicines11051484http://hdl.handle.net/2263/95220DATA AVAILABILITY STATEMENT : All data created or analyzed during this study are available from the corresponding author upon request.SUPPLEMENTARY MATERIALS : FIGURE S1: Dose–response curves showing the alteration of cell density, N = 5 biological repeats (A) and APH, N = 4 biological repeats (B) of monolayers treated with half-log dilutions of 32 M doxorubicin for 72 h. The IC50 was calculated using a non-linear regression curve fit (log[inhibitor] vs. response) with a robust fit.Three-dimensional cell culture models are increasingly adopted as preferred pre-clinical drug testing platforms, as they circumvent limitations associated with traditional monolayer cell cultures. However, many of these models are not fully characterized. This study aimed to characterize a BT-20 triple-negative breast carcinoma spheroid model and assess its susceptibility to doxorubicin in comparison to a monolayer model. Spheroids were developed using the liquid overlay method. Phenotypic attributes were analyzed by characterizing changes in size, gross morphology, protein content, metabolic activity, hypoxic status, and cell–cell junctions. The cytotoxic range of doxorubicin in monolayers was determined using the sulforhodamine B assay, and the comparative effect of toxic and sub-toxic concentrations was assessed in both spheroids and monolayers. Similar to the in vivo microenvironment, spheroids had a heterogeneous spatial cytoarchitecture, inherent hypoxia and strong adherens junctions. Doxorubicin induced dose-dependent cytotoxicity in monolayers (IC25: 130 nM, IC50: 320 nM and IC75: 1580 nM); however, these concentrations did not alter the spheroid size or acid phosphatase activity. Only concentrations 6 M had any effect on spheroid integrity. In comparison to monolayers, the BT-20 spheroid model has decreased sensitivity to doxorubicin and could serve as a better model for susceptibility testing in triple-negative breast cancer.en© 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.BT-20 CellsDoxorubicinDrug susceptibilityMulticellular spheroidsTriple-negative breast cancerTwo-dimensional culture modelThree-dimensional culture modelSDG-03: Good health and well-beingArticle