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Fig. 1 Medullary thyroid carcinoma (MTC) primary cells can be cultivated as multicellular spheroids enriched in stem-like cells. (A) representative images of two weeks old multicellular spheroids showing the morphological differences among patients. Scalebar 200 µm. (B) quantification of spheroids diameter growth during three weeks culture time. A significative increase was detected for all patients. 100 spheroids from 4 technical replicates were analyzed for each patient. (C, D) western blots images and densitometric quantification showing SOX2, OCT4, HIF2α and CD133 (stem markers), FOXA1 and TBB3 (neuroendocrine progenitors markers), p53 (proliferation, differentiation and reprogramming marker), E-cadherin and Vimentin (epithelial and mesenchymal markers, respectively) in the primary MTC tissue (T), tissue cellular dispersion (D) and PDS at 1, 2, and 3 weeks of culture. Each dot represents a single patient. R.U., relative units. n = 8. (E, F) confocal microscopy images and relative quantification of stem markers SOX2, OCT4, HIF2α, CD133 and neuroendocrine progenitors markers FOXA1 and TBB3 in MTC tissues bulk cellular tumor (BT) vs. perivascular areas (PV), known niches enriched in cancer stem-like cells. Insets highlight BT and PV areas at higher magnification. Vessels are indicated by asterisks in PV insets. Scalebar 100 µm. n = 12. Data represented as box and whiskers with 5–95 percentile range (B), mean ± SEM (D) and paired dotblot (F). Statistical analysis: One-way ANOVA followed by Tukey’s Multiple Comparison Test (B, D); paired t-test (F).