Search

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 S7. (A) Two-way unsupervised hierarchical clustering of RPPA data of 2 G1, 7 G2, 8 G3, and 3 G4 ccRCC populations for the expression of stem cell markers reported as heatmap. (B) mRNA expression of SOX2 gene in G2, G3, and G4 ccRCC samples as assessed by RT-qPCR. Mean of three independent experiments is reported. RRN18S was used as endogenous control. *p

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 S10. (A) Kaplan-Meier curve for the Disease Free Survival (DFS) in the engrafted (Engraf) and not engrafted (No Engraf) groups. (B) Histogram showing FACS analysis results of a PDX derived from a G4 metastatic patient with a sarcomatoid phenotype at esordium for the expression of selected markers. (C) Luciferase analysis representative image of G4 ccRCC injected mice (seven mice/group) and after 21Â days of treatment with Sunitinib by IVIS imaging. (D) Histogram showing luciferase photon emission of the vehicle and Sunitinib treated mice at days 0 and 21 of treatment and evaluated by IVIS imaging. (E) Representative Haematoxylin and Eosin staining of tumor sections from treated mice. Microscope used Nikon Eclipse 55i, magnification 10X (upper panel) and 20X (lower panel) (F) Two-way unsupervised hierarchical clustering of 20 samples for the expression of endpoints representing the most common drug targets. Green arrows represent recurrent patients, while the black arrow represents a metastatic (M1) sample at esordium. (PDF 542 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 S9. Hematoxylin and Eosin staining of PDXs versus parental primary tumor. G4 tumors often retain both epithelial and sarcomatoid phenotypes. Xenografts are frequently representative of most aggressive parental part. Representative images report epithelial and sarcomatoid phenotype belonging to the same patient. Xenograft image mirrors parental tumor aggressive phenotype. (PDF 114 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)

Context. One of the characteristic findings of intraductal papillary mucinous neoplasms (IPMN) is the presence of a direct communication between the lesion and the ductal pancreatic system and when magnetic resonance cholangiopancreatography (MRCP) shows uncertain findings, it is useful to perform a MRCP after secretin stimulation (MRCP-S) which provides a better visualization of the ductal system. Case Report. We present a case of 51-year-old man in whom, during a CT follow-up for a renal tumour, was found a cystic lesion of the pancreas. To better evaluate the lesion and its suspected communication with the pancreatic system, MR with gadolinium and MRCP and MRCP-S were performed. With the MRCP and MRI it was not possible to identify a clear communication between the cystic lesion and the ductal system. MRCP-S showed an increase in signal intensity of the lesion and its communication with the ductal system, allowing us to classify the cystic lesion as a main duct in intraductal papillary mucinous neoplasm. The patient underwent a surgical duodenal pancreatectomy. The histological result of the specimen confirmed the diagnosis of adenocarcinoma IPMN. Conclusion. In this case MRCP-S has allowed a clearer identification of the cystic lesion allowing a correct diagnosis and treatment.

Purpose Methods and Materials Results Conclusion References Personal Information, Purpose: INTRODUCTION: During the past year an increase of cystic pancreatic lesions, due to the widespread use of cross-sectional imaging, was observed. A correct assessment of pancreatic cystic lesions is essential as the different degree of malignancy of...

Purpose Methods and Materials Results Conclusion References Personal Information, Purpose: INTRODUCTION The accurate and early identification of hepatocellular carcinoma (HCC) is critical for planning the most appropriate therapeutic approach. The presence on a 4-phase multidetector CT scan or dynamic contrast enhanced MRI of a nodule...