Fig. 2

Adherent MTC cell lines recapitulate MTC tissues characteristics. (A) OncoPrint of the driver mutations and Variant of Unknown Significance (VUS), classified as Oncogenic Supporting, detected in MTC tissues and relative cell lines. All genetic alterations present in tissues were maintained in the respective cell lines. CCH, C-cell hyperplasia, SMTC, sporadic MTC, MEN2A, multiple endocrine neoplasia 2 A. Sample order: Tissue1, MTC1, Tissue2, MTC2, Tissue4, MTC4, Tissue5, MTC5, Tissue6, MTC6, Tissue7, MTC7, Tissue8, MTC8, Tissue9, MTC9. (B) Bright field images of crystal violet-stained MTC lines showing prevalent epithelial-like morphology, and eventual presence of fibroblast-like and squamous-like morphologies, indicative of contamination from other cell types than MTC. Scalebar 30 µm. (C) confocal microscopy images and quantification of different cell types present in the MTC lines, at early and late passages, defined as MTC cells (FOXA1+), fibroblasts (TE-7+), thyrocytes (EpCAM+, CD31−), and endothelial cells (EpCAM+, CD31+), indicative of significant contamination by fibroblasts, thyrocytes, and/or unidentified cells in three cell lines. n = 5. Scalebar 20 µm. (D, E) C-cells and MTC marker Calcitonin (CT) and neuroendocrine and C-cells progenitors marker Chromogranin A (CgA) secretion in the MTC lines cultivated in various differentiation conditions, being from more stem-promoting to more differentiation promoting one: sphere media (SM), adherent media (AM), adherent media with 10% FBS (AF) and complete F12K medium (F12K). 8 out of 9 cell lines secreted significant amounts of Calcitonin and Chromogranin A in differentiation promoting conditions, indicative of maintenance of MTC original characteristics. Each dot represents a different cell line. TT cells reference values are represented as gray diamonds and not included in the statistical analysis. n = 8. (F) Calcitonin and Chromogranin A mean levels of all cell lines at different passages in complete F12K medium, showing the maintenance of the secretive abilities during continuous culture up until p14. Each dot represents the median of all different cell lines. n = 8. (G) Epithelial thyroid marker Thyroglobulin (Tg) secretion in the MTC lines cultivated in various differentiation conditions, ranging from more stem-promoting to more differentiation-promoting one: sphere media (SM), adherent media (AM), adherent media with 10% FBS (AF) and complete 6H medium (6H), confirming the presence of thyrocytes contamination in two cell lines. Each dot represents a different cell line. TT cells reference values are represented as gray diamonds and not included in the statistical analysis. n = 8. Data represented as mean ± SEM (C), median ± IQR (D–G). Statistical analysis: Kruskal–Wallis test followed by Dunn’s Multiple Comparison Test (D, E, G, I).