Fig. 3

Generation and phenotypic characterization of CTCDOs cultures. A Time course images of CTCDOs culture. B Images of paraffin embedded sections obtained from CTCDOs (upper panels) and XDOs (lower panels). From left to right: Differential interference contrast (DIC), H&E staining, cytokeratin-20 (CK20), cytokeratin-7 (CK7), Caudal Type Homeobox 2 (CDX2). C Flow cytometry experiments showing the percentage of EpCAM, Vimentin (VIM), CK20 and CD44v6 positive cells in CTCDOs (purple bars) and XDOs (red bars) cultures. Values are obtained as the ratio between positive cells and total cells multiplied by 100, and represent the mean ± SD of three independent experiments performed with technical replicates. D Percentage of EpCAM⁺/CD44v6⁺, VIM⁺/CD44v6⁺ cells in CTCDOs (purple bars) and XDOs (red bars). Values are obtained as the ratio between positive cells and total cells multiplied by 100, and represent the mean ± SD of three independent experiments performed with technical replicates. E CTCs enumeration performed on the whole ScreenCell® filters obtained from each patient (Pt 1-6). Aggregates containing ≥3 CD45-negative cells were considered as CTCs clusters. Single cells: pink bar, clusters: violet bar. F CTCs isolated from the peripheral blood of CRC patients and stained with EpCAM (green)/CK20 (red); VIM (green)/CK20 (red); CD44v6 (green)/CK20 (red); (G) CTCs isolated from the peripheral blood of CRC patients and stained with CD44v6 (green)/EpCAM (yellow)/CK20 (red) or CD44v6 (green)/VIM (yellow)/CK20 (red). Nuclei were stained with DAPI. CD45 staining was performed with diaminobenzidine (DAB) and detected on differential interference contrast (DIC) images, appearing negative on CTCs and positive on blood cells (Additional file 6: Fig. S2D). Images are representative of at least three CTCs isolated from three different patients. Magnification 60x, 5x zoom. Bar 10 μM