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Additional file 1:. Supplementary Methods.

Additional file 11: Figure S5. ZEB2 expression in TNM stages, correlation with RFS and CMS in stage 2 CRC patients. ZEB2 transcript levels in the indicated number of CRC patients across all TNM stages. One-way ANOVA resulted in non-significant differences between stages. Outliers are depicted as crosses. n = 1079.

Additional file 8: Figure S2. Expression of pCRAF in vivo and in vitro and complementary RPPA data analysis. a Representative confocal microscopy images of PKH26-positive areas (yellow) in xenograft sections immunostained with anti-pCRAF S338 (green) and PROMININ1 (red). Scale bar, 80 μm. b Representative confocal microscopy images of SW480 cells treated for 48 h with 10 μM etoposide or 10 μM irinotecan and stained with anti-pCRAF S338 antibody. Scale bar, 20 μm. c Spatial representation of principal component (PC) analysis computed on a matrix having loading values of the two components, Factor 1 and Factor 2 that discriminates among PKH26+ and PKH26− samples. Results obtained on three PKH26+ versus PKH26− samples, n = 3 pools of 12 tumors each. d Spatial representation of scores of components representing relative RPPA antibodies values (Factor 1 and Factor 2). Results obtained on three PKH26+ versus PKH26− samples, n = 3 pools of 12 tumors each.

Additional file 10: Figure S4. In vivo effects of ZEB2 overexpression in CCSCs. a Volume of xenografts derived from CCSCs transduced with pLenti-GFP (Vector, black line/triangles) or with pLenti-GFP-ZEB2 (ZEB2, red line/squares). Graph shows the mean ± SEM, 6 tumors/group. **P

Additional file 9: Figure S3. Trends of ZEB2 overexpression in cultured cells. a Percentage of GFP positivity in CCSCs (diamonds) or SW480 (circles) cells transduced with empty pLenti-GFP (Vector) or with pLenti-GFP-ZEB2 (ZEB2) as assessed by flow cytometry for 5 weeks following lentiviral transduction and sorting (day 0). Graph shows the mean ± SD of three independent experiments. b Representative confocal image of CCSCs and SW480 cells transduced with GFP-ZEB2 and labeled with anti-Ki67 at day 28 after sorting. Circles indicate rare ZEB2+ cells, which are also Ki67−. Scale bar, 50 μm.

Additional file 4: Figure S1. Chemoresistance and marker expression of PKH26-positive cells. a Representative flow cytometry analysis of SW480 cells stained with PKH26 and treated with 2,5 μM oxaliplatin (OXA) starting from day 11, as described in Fig. 1a. Cell viability plots obtained with 7-AAD staining are shown below PKH26 plots, and the percentage of viable cells is indicated below each plot. b Representative flow cytometry analysis of EpCAM and PKH26 on xenograft-derived CCSCs 3 weeks upon subcutaneous injection of PKH26-stained cells in NSG mice. IgG, isotype control antibody. c Representative flow cytometry analysis of PKH26 and Ki67 in xenograft-derived EpCAM+ CCSCs at 3 weeks of tumor growth. d Representative flow cytometry analysis of PKH26 and PROMININ1 in xenograft-derived EpCAM+ CCSCs at 3 weeks of tumor growth.

Figure S1. Metabolites plasma concentrations (MPR and oxo-4-HPR) determined in mice after single oral treatment of 4 doses of Bio-nFeR: 10, 50, 100 and 200 mg/Kg. The analysis was performed after 0, 24, 48 h. Figure S2. Evaluation of the 50% inhibitory concentration (IC50) of BionFeR and NCI-FeR formulation was done on a representative lung cancer stem cell line (LCSC). The cells were exposed to the indicated drug doses (0–30 μM) and cell viability was evaluated by CellTiter-Glo after 72 h. Figure S3. A) Sphingomyelin-Ceramide (SM-CER) and Sphingomyelin- Dihydro-Ceramide (SM-DH-CER) content in lung (LC), melanoma (MEL) and colon (CRC) tumors treated with Bio-nFeR at single 100–150 mg/Kg administration were quantified through Ultra High Performance Liquid Chromatography (UHPLC), normalized to control and plotted as fold change. B) Quantification of Glucosyl-Ceramide (GLC-CER) and Glucosyl-Dihydro-Ceramide (GLC-DH-CER) in the same samples as in A, normalized to control and plotted as fold change. Figure S4. Cancer stem cell marker expression in CSC-culture conditions and in CSC-derived xenografts. A) Flow cytometry analysis of Aldefluor in (left panels) and CD44v6 (middle and right panels) in the indicated CSC and xenograft samples. B) Immunoblot analysis of CSC antigens c-Kit and SOX-2 in the indicated control (−) or BionFeR treated (+) melanoma xenografts. β-actin blot was used for equal loading control. Figure S5. Growth curves of lung CSC-derived xenografts in control mice or mice treated with Bio-nFeR or NCI-FeR at 100 mg/kg dose for the indicated times. Mean ± standard error is shown. *P