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Table S1: Primer sequences for SYBR Green RT-qPCR. (PPTX 68 kb)

Table S3. Detailed characteristics of 28 VHL-WT-RCC patients. (PPTX 71 kb)

Table S2. Detailed characteristics of 23 VHL-MUT-RCC patients. (PPTX 71 kb)

The chemokine receptor CXCR4 is overexpressed and functional in colorectal cancer. To investigate the role of CXCR4 antagonism in potentiating colon cancer standard therapy, the new peptide CXCR4 antagonist Peptide R (Pep R) was employed. Human colon cancer HCT116 xenograft-bearing mice were treated with chemotherapeutic agents (CT) 5-Fluorouracil (5FU) and oxaliplatin (OX) or 5FU and radio chemotherapy (RT-CT) in the presence of Pep R. After two weeks, CT plus Pep R reduced by 4-fold the relative tumor volume (RTV) as compared to 2- and 1.6-fold reductions induced, respectively, by CT and Pep R. In vitro Pep R addition to CT/RT-CT impaired HCT116 cell growth and further reduced HCT116 and HT29 clonal capability. Thus, the hypothesis that Pep R could target the epithelial mesenchyme transition (EMT) process was evaluated. While CT decreased ECAD and increased ZEB-1 and CD90 expression, the addition of Pep R restored the pretreatment expression. In HCT116 and HT29 cells, CT/RT-CT induced a population of CD133+CXCR4+ cells, supposedly a stem-resistant cancer cell population, while Pep R reduced it. Taken together, the results showed that targeting CXCR4 ameliorates the effect of treatment in colon cancer through inhibition of cell growth and reversal of EMT treatment-induced markers, supporting further clinical studies.

// Sara Santagata 1 , Maria Napolitano 1 , Crescenzo D'Alterio 1 , Sonia Desicato 2 , Salvatore Di Maro 3 , Luciana Marinelli 3 , Alessandra Fragale 4 , Maria Buoncervello 4 , Francesco Persico 5 , Lucia Gabriele 4 , Ettore Novellino 3 , Nicola Longo 5 , Sandro Pignata 2 , Sisto Perdona 2 and Stefania Scala 1 1 Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”-IRCCS, 80131 Naples, Italy 2 Uro-Gynecological Department, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”-IRCCS, 80131 Naples, Italy 3 Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy 4 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita, 00161 Rome, Italy 5 Urology Division, University Federico II, 80131 Naples, Italy Correspondence to: Stefania Scala, email: s.scala@istitutotumori.na.it , scalaste@gmail.com Keywords: CXCR4, immune suppression, renal cell carcinoma, T regulatory cells, tumor microenvironment Received: May 07, 2017 Accepted: June 25, 2017 Published: August 19, 2017 ABSTRACT With the intent to identify biomarkers in renal cell carcinoma (RCC) the functional status of T-regulatory cells (Tregs) was investigated in primary RCC. Tregs were isolated from tumoral-(TT), peritumoral tissue-(PT) and peripheral blood-(PB) of 42 primary RCC patients and function evaluated through effector T cells (Teff) proliferation, cytokines release and demethylation of Treg Specific Region (TSDR). The highest value of Tregs was detected in TT with the uppermost amount of effector-Tregs-(CD4 + CD25 hi FOXP3 hi CD45RA - ). PB-RCC Tregs efficiently suppress Teff proliferation compared to healthy donor (HD)-Tregs and, at the intrapatient evaluation, TT-derived Tregs were the most suppressive. Higher demethylation TSDR was detected in TT- and PB-RCC Tregs vs HD-Tregs ( P

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.

// Davide Gentilini 1 , Stefania Scala 2 , Germano Gaudenzi 3 , Paolo Garagnani 4, 5 , Miriam Capri 4, 5 , Matteo Cescon 6 , Gian Luca Grazi 7 , Maria Giulia Bacalini 8 , Serena Pisoni 1 , Alessandra Dicitore 1 , Luisa Circelli 9 , Sara Santagata 2 , Francesco Izzo 10 , Anna Maria Di Blasio 1 , Luca Persani 1, 3 , Claudio Franceschi 4, 5, 8 and Giovanni Vitale 1, 3 1 Istituto Auxologico Italiano IRCCS, Cusano Milanino, Milan, Italy 2 Functional Genomics, Istituto Nazionale per lo Studio e la Cura dei Tumori, IRCCS Fondazione “G. Pascale”, Napoli, Italy 3 Department of Clinical Sciences and Community Health (DISCCO), University of Milan, Milan, Italy 4 Department of Experimental, Diagnostic and Specialty Medicine, Alma Mater Studiorum, University of Bologna, Bologna, Italy 5 Interdepartmental Center "L. Galvani", University of Bologna, Bologna, Italy 6 DIMEC-Department of General Surgery and Medicine Sciences, S. Orsola-Malpighi Hospital, Bologna, Italy. 7 Regina Elena National Cancer Institute Via Elio Chianesi 53, Rome, Italy 8 IRCCS Institute of Neurological Sciences, Bologna, Italy 9 Department of Experimental Oncology, Laboratory of Molecular Biology and Viral Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione “G. Pascale”, Napoli, Italy 10 Department of Surgical Oncology, Abdominal and Hepatobiliary Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, IRCCS Fondazione “ G. Pascale”, Napoli, Italy Correspondence to: Giovanni Vitale, email: giovanni.vitale@unimi.it Keywords: hepatocellular carcinoma, stochastic epigenetic mutation, epigenetics, DNA methylation, genome-wide Received: January 27, 2017 Accepted: April 15, 2017 Published: April 27, 2017 ABSTRACT Hepatocellular carcinoma (HCC) results from accumulation of both genetic and epigenetic alterations. We investigated the genome-wide DNA methylation profile in 69 pairs of HCC and adjacent non-cancerous liver tissues using the Infinium HumanMethylation 450K BeadChip array. An innovative analytical approach has been adopted to identify Stochastic Epigenetic Mutations (SEMs) in HCC. HCC and peritumoral tissues showed a different epigenetic profile, mainly characterized by loss of DNA methylation in HCC. Total number of SEMs was significantly higher in HCC tumor (median: 77,370) than in peritumoral (median: 5,656) tissues and correlated with tumor grade. A significant positive association emerged between SEMs measured in peritumoral tissue and hepatitis B and/or C virus infection status. A restricted number of SEMs resulted to be shared by more than 90% of HCC tumor samples and never present in peritumoral tissue. This analysis allowed the identification of four epigenetically regulated candidate genes (AJAP1, ADARB2, PTPRN2, SDK1), potentially involved in the pathogenesis of HCC. In conclusion, HCC showed a methylation profile completely deregulated and very far from adjacent non-cancerous liver tissues. The SEM analysis provided valuable clues for further investigations in understanding the process of tumorigenesis in HCC.

A neoadjuvant clinical trial was previously conducted in patients with resectable colorectal cancer liver metastases (CRLM). At a median follow up of 28 months, 20/33 patients were dead of disease, 8 were alive with disease and 5 were alive with no evidence of disease. To shed further insight into biological features accounting for different outcomes, the expression of CXCR4-CXCL12-CXCR7, TLR2-TLR4, and the programmed death receptor-1 (PD-1)/programmed death-1 ligand (PD-L1) was evaluated in excised liver metastases. Expression profiles were assessed through qPCR in metastatic and unaffected liver tissue of 33 CRLM neoadjuvant-treated patients. CXCR4 and CXCR7, TLR2/TLR4, and PD-1/PD-L1 mRNA were significantly overexpressed in metastatic compared to unaffected liver tissues. CXCR4 protein was negative/low in 10/31, and high in 21/31, CXCR7 was negative/low in 16/31 and high in 15/31, CXCL12 was negative/low in 14/31 and high in 17/31 CRLM. PD-1 was negative in 19/30 and positive in 11/30, PD-L1 was negative/low in 24/30 and high in 6/30 CRLM. Stromal PD-L1 expression, affected the progression-free survival (PFS) in the CRLM population. Patients overexpressing CXCR4 experienced a worse PFS and cancer specific survival (CSS) (p = 0.001 and p = 0.0008); in these patients, KRAS mutation identified a subgroup with a significantly worse CSS (p < 0.01). Thus, CXCR4 and PD-L1 expression discriminate patients with the worse PFS within the CRLM evaluated patients. Within the CXCR4 high expressing patients carrying Mut-KRAS in CRLM identifies the worst prognostic group. Thus, CXCR4 targeting plus anti-PD-1 therapy should be explored to improve the prognosis of Mut-KRAS-high CXCR4-CRLMs.

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.