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Additional file 1: Figure S1. Extended data for figure 3 and 4. Uncropped Western blot images showing A PDGFR-β and related actin; B cleaved caspase-3 and related actin; C eNOS and VEGF with related actin; D Tie2, ANGPT1 and related actin. Boxed areas correspond to cropped regions shown in figure 3C and 3E. Specific target bands were selected according to the molecular weight reported in antibodies’ datasheets. Table S1: raw RPPA data.

Additional file 1:. Supplementary Methods.

Additional file 1: Supplementary Table 1. List of drugs used for small-molecule kinase inhibitor screening (10 mM in DMSO). Supplementary Table 2. List of antibodies used for Reverse-Phase Protein microArrays (RPPA) analysis. Supplementary Table 3. Patient and GSC line characteristics. Supplementary Table 4. List of genes corresponding to significant antibodies and grouped using the Venn diagram in Fig. 4C. Supplementary Figure S1. A-D. Morphological changes of the four GSC lines used in the study (A, GSC#1; B, GSC#61; C, GSC#83; D, GSC#163) after being induced to transdifferentiate for 2 weeks. Left panel, tumorspheres in stem cell medium; right panel, net-like structures under endothelial conditions (magnification 10X). Supplementary Figure S2. (A) Fluorescent-activated cell sorting dot plots of CD34−/low and CD34high GSC#163 after two weeks of culture in endothelial conditions under hypoxia. Percentage and squares indicate the sorted subpopulations of cells with different CD34-expression levels (left, IgG1 isotype control sample; right, CD34 sample). (B-C) Immunohistochemical analysis of CD34low (B) and CD34high (C) GdEC subcutaneous tumor xenografts based on the expression of the astrocytic marker glial fibrillary acidic protein (GFAP, right panels), showing tumors with different levels of differentiation. (Left panels, haematoxylin and eosin staining; magnification 200X). Supplementary Figure S3. Concentration-response assays on U87MG and all the four glial cell lines derived from the selected GSC lines. Supplementary Figure S4. Cytofluorimetric cell-by-cell analysis of viability in four different GSC lines treated with 10, 100, or 1000 nM elesclomol in the presence or absence of the following cell death inhibitors: z-VAD, necrostatin-1, ferrostatin-1, 3-MA, NAC, and CoQ at the indicated concentrations. Results obtained from four independent experiments are expressed as percentage vs control untreated cells and reported as means ± SD. Supplementary Figure S5. Cytofluorimetric cell-by-cell analysis of cell viability (A), mitochondrial ROS production (B), mitochondrial membrane potential (C), and GSH (D) in four different GSC lines treated with 10, 100, or 1000 nM elesclomol in the presence or absence of the copper chelating agent TTM. Results obtained from four independent experiments are expressed as percentage vs control untreated cells and reported as means ± SD. Supplementary Figure S6. Cytofluorimetric cell-by-cell analysis of cell viability, mitochondrial ROS production, mitochondrial membrane potential, and GSH in HMVECs, used as a control of nontumoral endothelial cell line, treated with 10, 100, or 1000 nM Elesclomol in the presence or absence of the copper chelating agent TTM. Results obtained from four independent experiments are expressed as percentage vs control untreated cells and reported as means ± SD. Supplementary Figure S7. Illustration of the rationale suitable for the choice of rank k, a critical parameter that defines the number of metagenes used to approximate the target matrix (Gaujoux & Seoighe, 2010). A) Measurements are applied to both real data (circles) and randomized data (triangles). The rationale for choosing rank stems on diverse metrics, i) trend of the cophenetic coefficient: Brunet et al. (2004) suggest choosing the smallest value of k for which there is a decrease in the trend of the cophenetic; ii) trend of the dispersion coefficient introduced by Kim & Park. (2007); iii) explained variance by increasing rank; iv) trend of residuals; v) trend of RSS: Hutchins et al. (2008) suggest taking the first rank value for which we have an inflection point. Frigyesi et al. (2008) instead consider the first rank value for which the decrease of the RSS on real data is less than the decrease of the RSS on the random data; vi) silhouette values measured on the matrices of the base, of the coefficients and the consensus matrix; vii) trend of the sparseness introduced by Hoyer (2004). B) Multiple consensus maps corresponding to different value of k. Supplementary Figure S8. Heatmap of the most important antibodies in each of the k = 6 metagenes resulting from the model. Supplementary Figure S9. Principal Component Analysis (PCA) algorithm applied to Elesclomol data, whereby each cell line is considered as a function of the antibodies. A) Scree plot. Given the low amount of variance explained by the variables above the fifth, we considered up to 5 principal components. B) Biplots using cell lines and growth conditions as scores. Ellipses represent the 95% probability of finding sample score values. Supplementary Figure S10. Principal Component Analysis (PCA) biplots of components of the antibodies using (A) Time and (B) treatment, respectively. Ellipses represent the 95% probability of finding sample score values. Supplementary Figure S11. Concent ration-response assays on all the four selected GSC lines for setting the dose of Elesclomol most suitable for the combination with TMZ.

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.

Figure S1. Additional RPPA endpoints. (PDF 89474 kb)

Table S1. Complete list of antibodies used for RPPA analysis and their main related information. (PDF 26 kb)

Figure S2. Clonogenic Assay. (PDF 440 kb)

Figure S6. (A) Freshly dissociated tissues maintained for one week in serum-free stem cell-isolating medium supplemented with Epidermal Growth Factor (EGF), basic Fibroblast Growth Factor (b-FGF), DMEM (Dulbecco Modified Eagle Medium), or Glutamine and FBS (Fetal Bovine Serum) supplemented medium, and analyzed by cytofluorimetric analysis. CD45 (PE-Cy7), CD146 (PE), CD44 (H450-Pacific Blue) and EpCAM (FITC) antigens were analyzed. TOPRO3 was used for gating vital cells. (B) The histograms report growth rate fold change of cells described in A, 4 and 10 days after sorting. Control represents (red dashed line) value = 1 i.e. reference relative count at sorting and plating day. Mean of three independent experiments is reported. Values are mean ± s.d (C) Colony forming assay of EpCAM+/CD146+/CD44+ and triple negative sorted cells and non-sorted population maintained in culture one week in stem serum free medium (D) Mean colony size of EpCAM+/CD146+/CD44+ and triple negative sorted cells and non-sorted population maintained in culture one week in stem serum free medium. Mean of three independent experiments is reported. Values are mean ± s.d. (PDF 231 kb)

Figure S8. (A) Representative images of Western blot analysis of pAKT S473 and pERK T202/Y204 proteins in clear renal cancer isolated cells (G3 and G4) and commercial lines [primary tumor 786–0 (786) and metastatic Caki-1 (CK1)]. GAPDH expression was used as internal control (B) Anti-mTOR and VEGFR2 protein staining in two representative samples classified by (ISUP) grading as G3 and G4 cases by immunohistochemistry assay. Microscope used Nikon Eclipse 55i, magnification 20X. (C) Representative images of Western blot analysis of pAKT S473 and pERK T202/Y204 proteins in non-sorted stem serum free clear renal cancer enriched cells (Stem Tot.), EpCAM+/CD146+/CD44+ (Stem+) and triple negative (Stem-) sorted cells vs non-sorted clear renal cancer cells maintained in DMEM-FBS condition (DMEM Tot) and evaluated one week after culture. (D) Two-way unsupervised hierarchical clustering of 18 ccRCC samples for the expression of proteins belonging to the angiogenesis pathway. Highlighted in the yellow box are overexpressed protein commonly shared in samples of patients that underwent progression (red arrows). N1 and M1 samples were excluded from the analysis (E) Receiver operating characteristic (ROC) curve showing sensitivity and specificity of HIF-1 alpha and phospho-mTOR (S2448) protein RPPA expressions in predicting progression. The true positive rate (sensitivity) is plotted in function of the false positive rate (100-specificity). The area under the ROC curve (AUC) represents a measure of how well the HIF-1 alpha and phospho-mTOR (S2448) protein RPPA expressions distinguishes progression group from no progression [0.96 (p

Figure S5. Freshly dissociated tissues were maintained three days in serum-free stem cell-isolating medium supplemented with Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (b-FGF). On the left a representative image of the sorting of EpCAM+/CD146+/CD44+ populations (EpCAM+/CD146+/CD44+) and triple negative (EpCAM-/CD146-/CD44-) by FACS ARIA cytometer was reported. Images of colonies of both sorted sub-populations were reported on the right. Yellow and pink boxes mirror cytometer density plot. Pink dashed line represents matrigel front of cell invasion. (PDF 179 kb)

Figure S3 (A) RPPA-TCGA elaboration of E-Cadherin and Fibronectin expressions. Data were obtained from macrodissected clear cell renal cancer tissues (GDC-database- https://tcga-data.nci.nih.gov/docs/publications/kirc_2013/ ) and reported for grading, stage and for progression rate by RPPA. (B) mRNA level elaboration of EpCAM, CD146(MCAM) and CD44 antigens. Data were obtained from GSE48550 microarray and were analyzed on different kinds of renal stem cells. (C) TOPRO3 staining for cell viability evaluation of populations maintained for three days (upper panels) and one week (Lower panels) in serum-free stem cell-isolating medium supplemented with Epidermal Growth Factor (EGF), basic Fibroblast Growth Factor (b-FGF), DMEM (Dulbecco Modified Eagle Medium), Glutamine and FBS (Fetal Bovine Serum) supplemented medium and evaluated by cytofluorimetric analysis. Blue and Black areas represent vital and dead cells respectively. (PDF 389 kb)

Table S2. Clinical features of 20 collected ccRCC patients including: 2 G1; 7 G2; 8 G3 and 3 G4 processed by RPPA. (PDF 488 kb)

Figure S4. (A) Fresh dissociated tissues maintained for three days in serum-free stem cell-isolating medium supplemented with Epidermal Growth Factor (EGF), basic Fibroblast Growth Factor (b-FGF), DMEM (Dulbecco Modified Eagle Medium), or in Glutamine and FBS (Fetal Bovine Serum) supplemented medium, and analyzed by cytofluorimetric analysis. CD45 (PE-Cy7), CD146(PE), CD44 (H450-Pacific Blue) and EpCAM(FITC) antigens were analyzed. TOPRO3 was used for gating vital cells. (B-C) Images and clonogenic population percentage of cells maintained in both conditions after three days of culture by Colony forming assay. Colonies distinguished on the basis of their shape in the two conditions: spheroidal (blue box) and bidimensional (red box). (D) Percentage of colonies distinguished on the basis of their shape in the two conditions was reported: spheroidal (blue box) and bidimentional (red box) such as in B. (PDF 229 kb)

Table S3. List of total and phosphorylated proteins analyzed by RPPA. In blue, positively expressed proteins. (PDF 87 kb)

Figure S1. (A) (PANEL1) Table of 1286 ccRCC patient distribution: 1013 tumor free patients at 36 months from surgery, 130 metastatic (M1) patients at diagnosis time and 143 recurrent patients at 36 months after surgery were reported. (PANEL2) Table of 57 ccRCC cancer patient distribution: 37 tumor free patients at 36 months from surgery, 6 metastatic (M1) patients at diagnosis time and 14 recurrent patients at 36 months from surgery were reported. (B) Representative immunofluorescence of DAPI-stained tumor derived spheroids. (C) Representative image of 7- aminoactinomycin D staining (7AAD) of in vitro isolated populations by flow cytometry. (D) Table reporting distribution of specific antigen expression percentages (%) in all studied ccRCC populations. (E) Representative images of flow cytometry analysis showing the expression of the epithelial and undifferentiated cell markers EpCAM, CD24, CD10, CD90, CD44 and CD146 mesenchymal stem cell markers in ccRCC isolated populations. Background staining was calculated by using appropriate isotype controls. (F) Flow cytometry analysis of cell lines 786–0 and Caki-1 representative of primary and metastatic tumor, respectively. One representative staining of three independent experiments is shown. (PDF 243 kb)