Use of in vivo phage display to engineer novel adenoviruses for targeted delivery to the cardiac vasculature

We performed in vivo phage display in the stroke prone spontaneously hypertensive rat, a cardiovascular disease model, and the normotensive Wistar Kyoto rat to identify cardiac targeting peptides, and then assessed each in the context of viral gene delivery. We identified both common and strain-selective peptides, potentially indicating ubiquitous markers and those found selectively in dysfunctional microvasculature of the heart. We show the utility of the peptide, DDTRHWG, for targeted gene delivery in human cells and rats in vivo when cloned into the fiber protein of subgroup D adenovirus 19p. This study therefore identifies cardiac targeting peptides by in vivo phage display and the potential of a candidate peptide for vector targeting strategies

A complex of α(6) integrin and E-cadherin drives liver metastasis of colorectal cancer cells through hepatic angiopoietin-like 6.

Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and α(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural α(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC

Robotic-assisted radical cystectomy: the first multicentric Brazilian experience

The objective of this study is to report the first multicentric Brazilian series and learning curve of robotic radical cystectomy (RARC) with related intra- and postoperative outcomes. We retrospectively analyzed 37 RARC prospectively collected at four different centers in Brazil, from 2013 to 2019. We analyzed the patient’s demographics, pathological tumor, and nodal status, as well as intra- and postoperative outcomes. Statistical analysis was performed with the IBM (SPSS version 25) software. Overall, 86% were male, and the median age was 69 years. 83% had muscle-invasive bladder cancer, and 17% a high-grade, recurrent non-muscle-invasive tumor. The median operative time was 420 min with 300 min as console time. Median blood loss was 350 ml and transfusion rate was 10%. In 68% of the cases, we performed an intracorporeal Bricker urinary diversion, 24% intracorporeal neobladder, and 8% ureterostomy. Six patients (16%) had a Clavien 1–2, 8% had Clavien 3, 2.5% had a Clavien 4, and 5% had Clavien 5. The median length of hospital stay was 7 days. The final pathological exam pointed out pT0 in 16%, pT1 in 8%, pT2 in 32%, ≥ pT3 in 27%, and 16% pTis. 95% had negative surgical margins. The survival at 30, 90, and 180 days was 98%, 95%, and 95%, respectively. To our knowledge, this is the first multicentric series of RARC reporting the learning curve in Brazil; even if still representing a challenging procedure, RARC could be safely and effectively faced by experienced surgeons at centers with high volumes of robotic surgery

A complex of α6 integrin and E-cadherin drives the liver metastasis of colorectal cancer cells by a physical and functional interaction with hepatic angiopoietin-like 6

Homing of colorectal cancer (CRC) cells to the liver is a non-random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis-specific peptide ligand (CGIYRLRSC) that binds a complex of E-cadherin and α(6) integrin on the surface of CRC cells. We identify angiopoietin-like 6 protein as a peptide-mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin-like 6 and tumoural α(6) integrin/E-cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α(6) integrin and E-cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC

Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer

Aggressive variant prostate cancers (AVPC) are a clinically defined group of tumors of heterogeneous morphologies, characterized by poor patient survival and for which limited diagnostic and treatment options are currently available. We show that the cell surface 78-kDa glucose-regulated protein (GRP78), a receptor that binds to phage-display-selected ligands, such as the SNTRVAP motif, is a candidate target in AVPC. We report the presence and accessibility of this receptor in clinical specimens from index patients. We also demonstrate that human AVPC cells displaying GRP78 on their surface could be effectively targeted both in vitro and in vivo by SNTRVAP, which also enabled specific delivery of siRNA species to tumor xenografts in mice. Finally, we evaluated ligand-directed strategies based on SNTRVAP-displaying adeno-associated virus/phage (AAVP) particles in mice bearing MDA-PCa-118b, a patient-derived xenograft (PDX) of castration-resistant prostate cancer bone metastasis that we exploited as a model of AVPC. For theranostic (a merging of the terms therapeutic and diagnostic) studies, GRP78-targeting AAVP particles served to deliver the human Herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which has a dual function as a molecular-genetic sensor/reporter and a cell suicide-inducing transgene. We observed specific and simultaneous PET imaging and treatment of tumors in this preclinical model of AVPC. Our findings demonstrate the feasibility of GPR78-targeting, ligand-directed theranostics for translational applications in AVPC

Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these “side” effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.Fil: Canals, Daniel. Stony Brook University; State University of New York;Fil: Salamone, Silvia. Stony Brook University; State University of New York;Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Nemeth, Erika. Stony Brook University; State University of New York;Fil: Aguilar, Daniel. Biomedical Research Networking Center in Hepatic and Digestive Diseases; EspañaFil: Hernandez Corbacho, María José. Stony Brook University; State University of New York;Fil: Adada, Mohamad. Stony Brook University; State University of New York;Fil: Staquicini, Daniela I.. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Arap, Wadih. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Pasqualini, Renata. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Haley, John. Stony Brook University; State University of New York;Fil: Obeid, Lina M.. Stony Brook University; State University of New York;Fil: Hannun, Yusuf A.. Stony Brook University; State University of New York

Ceramide launches an acute anti-adhesion pro-migration cell signaling program in response to chemotherapy

Chemotherapy has been reported to upregulate sphingomylinases and increase cellular ceramide, often linked to the induction to cell death. In this work, we show that sublethal doses of doxorubicin and vorinostat still increased cellular ceramide, which was located predominantly at the plasma membrane. To interrogate possible functions of this specific pool of ceramide, we used recombinant enzymes to mimic physiological levels of ceramide at the plasma membrane upon chemotherapy treatment. Using mass spectrometry and network analysis, followed by experimental confirmation, the results revealed that this pool of ceramide acutely regulates cell adhesion and cell migration pathways with weak connections to commonly established ceramide functions (eg, cell death). Neutral sphingomyelinase 2 (nSMase2) was identified as responsible for the generation of plasma membrane ceramide upon chemotherapy treatment, and both ceramide at the plasma membrane and nSMase2 were necessary and sufficient to mediate these “side” effects of chemotherapy on cell adhesion and migration. This is the first time a specific pool of ceramide is interrogated for acute signaling functions, and the results define plasma membrane ceramide as an acute signaling effector necessary and sufficient for regulation of cell adhesion and cell migration under chemotherapeutical stress.Fil: Canals, Daniel. Stony Brook University; State University of New York;Fil: Salamone, Silvia. Stony Brook University; State University of New York;Fil: Santacreu, Bruno Jaime. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Nemeth, Erika. Stony Brook University; State University of New York;Fil: Aguilar, Daniel. Biomedical Research Networking Center in Hepatic and Digestive Diseases; EspañaFil: Hernandez Corbacho, María José. Stony Brook University; State University of New York;Fil: Adada, Mohamad. Stony Brook University; State University of New York;Fil: Staquicini, Daniela I.. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Arap, Wadih. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Pasqualini, Renata. Rutgers Cancer Institute of New Jersey; Estados UnidosFil: Haley, John. Stony Brook University; State University of New York;Fil: Obeid, Lina M.. Stony Brook University; State University of New York;Fil: Hannun, Yusuf A.. Stony Brook University; State University of New York

A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Gillson-Longenbaugh FoundationNational Institutes of Health (NIH/NCATS) through the NIH Common Fund, Office of Strategic Coordination (OSC)AC Camargo Canc Ctr, Lab Med Genom, Sao Paulo, SP, BrazilAC Camargo Canc Ctr, Lab Computat Biol, Sao Paulo, SP, BrazilUniv Sao Paulo, Inst Biomed Sci, Dept Cell & Dev Biol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Electron Microscopy Ctr, Sao Paulo, SP, BrazilUniv Texas MD Anderson Canc Ctr, Dept Expt Therapeut, Houston, TX 77030 USAUniv Texas MD Anderson Canc Ctr, Ctr RNA Interference & Non Coding RNAs, Houston, TX 77030 USAUniv New Mexico, Comprehens Canc Ctr, Albuquerque, NM 87131 USAUniv New Mexico, Sch Med, Div Hematol Oncol, Dept Internal Med, Albuquerque, NM 87131 USAUniv New Mexico, Sch Med, Div Mol Med, Dept Internal Med, Albuquerque, NM 87131 USARockefeller Univ, Lab Mol Immunol, 1230 York Ave, New York, NY 10021 USAFMUSP, Lab Neurociencias Alzira Denise Hertzog Silva LIM, Inst Psiquiatria, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Electron Microscopy Ctr, Sao Paulo, SP, BrazilFAPESP: 2011/09172-3FAPESP: 2014/26897-0Web of Scienc