Fig. 4

CTCDOs display increased aggressiveness, stem cell content and migratory ability. A Volume of subcutaneous tumor xenografts derived from XDOs (red line/squares) or CTCDOs (purple line/triangles). Mean ± SEM, 5 tumors per group. *P < 0.05 and **P < 0.01 by unpaired Student’s t test with Welch’s correction. B Representative images of immunofluorescence staining (40x magnification) of Bmi1, CK20 and CD133 in xenograft sections obtained from tumor xenografts of Fig. 4A; nuclei were stained with DAPI. C Quantification of Bmi1, CK20 and CD133 performed on xenograft sections obtained from tumor xenografts of Fig. 4A, 5 fields/section. D Self-renewal capacity of cells isolated from xenografts of Fig. 4A, evaluated as colony formation in semisolid culture and expressed as normalized colony size/percentage over plated cells. Values represent the mean ± SD of three technical replicates. P < 0.05; ***P < 0.001 by unpaired Student’s t test. E Single-cell assay performed with CTCDOs- and XDOs-derived cells (right) and time course images of CTCDOs and XDOs cultures (left). 10x magnification, bar 100 μm. Values represent mean ± SD of three independent experiments. **P < 0.01 by unpaired Student’s t test. F Tumor initiating cells content of CTCDOs and XDOs cultures was evaluated through serial transplantation/limiting dilution assays and quantified with the extreme limiting dilution analysis (ELDA) software. Five mice were used for each dilution point. ***P < 0.001. G Invasion/migration assay performed with CTCDOs and XDOs. The upper panel graph indicates the number of migrated CTCDOs and XDOs, while the lower panels show representative images of nuclei of CTCDOs- and XDOs migrated cells stained with DAPI (10x magnification). Values represent mean ± SD of three independent experiments. ***P < 0.001