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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)

Figure S2. (A) Hematoxylin and Eosin (H&E) and CD31 staining of formalin-fixed and paraffin- embedded (FPPE) of primary tumors. Three patients for each grading were analyzed. A representative image for samples is reported. (B) RPPA-TCGA elaboration of CD31 expression. 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. (C) Representative images of flow cytometry analysis showing the expression of the endothelial CD31, VE-Cadherin (VE-Cadh) and putative stem cell markers (CD133, CD105) in ccRCC isolated populations. The analysis was combined with CD44 expression. Background staining was calculated by using appropriate isotype controls. (PDF 402 kb)