Fig. 2: CDX-derived in vitro cell cultures are growing in both 2D and 3D conditions and are tumorigenic.

a Morphology of in vitro cultures established as spheroids (3D) and monolayer cell cultures (2D). Scale bar 200 μm. b In vivo tumorigenicity assay of established in vitro cultures. Cell culture grown as spheroids (3D), or monolayer (2D) was injected subcutaneously in NRG female mice, and tumours were measured weekly using a manual caliper. c Spheroid formation assay using in vitro 3D cell culture. Representative images from seeded 1000, 2500, and 5000 cells per well are presented. Scale bar 300 µm. d Quantification of spheroids’ growth from c presented as an average spheroid area in pixels. N = 22 wells per cell density. e Analysis of selected surface epithelial-like (EpCAM, CD9, CD24, CD49f, CD111), and mesenchymal-like (CD29, CD44, CD49c, CD97, CD113, integrin β5) markers using spectral flow cytometry. Results are presented as the mean percentage of cells positive for each marker from three independent repetitions. See Supplementary Fig. S3 for statistical analysis. f t-SNE plot showing pooled in vivo xenograft, and in vitro 2D and 3D cell culture from one representative repetition. See Supplementary Fig. S4 for additional clustering analyses. g Detection of breast cancer stem cell subpopulation CD44⁺/CD24⁻ in in vivo xenograft and in vitro cell cultures. h Quantification of CD44⁺/CD24⁻ breast cancer stem cells. Results are presented as mean ± SEM from 3 independent repetitions. Statistical analysis was performed using ordinary One-way ANOVA and Tukey´s multiple comparison test.