Memorial Sloan Kettering Cancer Center View Institution's Website 29 articles published in JoVE Cancer Research Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells Madelyn Espinosa-Cotton1, Hong-Fen Guo1, Nai-Kong V. Cheung1 1Department of Pediatrics, Memorial Sloan Kettering Cancer Center Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies. Cancer Research Surgical Technique for Superior Cervical Ganglionectomy in a Murine Model Qi Wang1, Chun-Hao Chen1, Hongbo Xu2, Sylvie Deborde1,3, Richard J. Wong1,3 1Department of Surgery, Memorial Sloan Kettering Cancer Center, 2Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, 3David M. Rubenstein Center for Pancreatic Cancer Research, Memorial Sloan Kettering Cancer Center The present protocol describes a mouse model of the ablation of adrenergic innervation by identifying and resecting the superior cervical ganglion. Cancer Research Combining Reflectance Confocal Microscopy with Optical Coherence Tomography for Noninvasive Diagnosis of Skin Cancers via Image Acquisition Ucalene Harris1, Milind Rajadhyaksha1, Manu Jain1 1Dermatology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center Here, we describe protocols for acquiring good-quality images using novel, noninvasive imaging devices of reflectance confocal microscopy (RCM) and combined RCM and optical coherence tomography (OCT). We also familiarize clinicians with their clinical applications so that they can integrate the techniques into regular clinical workflows to improve patient care. Biochemistry An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics Anthony Gaba*1, Hongyun Wang*1, Xiaohui Qu1 1Molecular Biology Program, Memorial Sloan Kettering Cancer Center Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation. Cancer Research In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System Manu Prasad*1,2, Sankar Jagadeeshan*1,2, Maurizio Scaltriti3, Irit Allon2,4, Moshe Elkabets1,2 1The Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, 2Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Human Oncology & Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center, 4Institute of Pathology, Barzilai University Medical Center Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations. Cancer Research Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses Kyrie J. Pappas1, Danielle Choi1, Charles L. Sawyers1,2, Wouter R. Karthaus1 1Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 2Howard Hughes Medical Institute Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Cancer Research Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting Chrysafis Andreou1, Anton Oseledchyk1, Fay Nicolson1, Naxhije Berisha1,2, Suchetan Pal1, Moritz F. Kircher1,3,4,5,6,7 1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Department of Chemistry, The Graduate Center of the City University of New York, 3Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, 4Center for Molecular Imaging and Nanotechnology (CMINT), Memorial Sloan Kettering Cancer Center, 5Gerstner Sloan Kettering Graduate School of Biomedical Sciences, 6Department of Radiology, Weill Cornell Medical College of Cornell University, 7Dana-Farber Cancer Institute and Harvard Medical Center Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology. Chemistry Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates Maria Davydova1, Guillaume Dewaele Le Roi1,2, Pierre Adumeau1, Brian M. Zeglis1,2,3,4 1Department of Chemistry, Hunter College of the City University of New York, 2Ph.D. Program in Chemistry, Graduate Center of the City University of New York, 3Department of Radiology, Memorial Sloan Kettering Cancer Center, 4Department of Radiology, Weill Cornell Medical College In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody. Chemistry Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction Rosemery Membreno1,2, Brendon E. Cook1,2,3, Brian M. Zeglis1,2,3,4 1Department of Chemistry, Hunter College of the City University of New York, 2Ph.D. Program in Chemistry, Graduate Center of the City University of New York, 3Department of Radiology, Memorial Sloan Kettering Cancer Center, 4Department of Radiology, Weill Cornell Medical College This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer. Cancer Research Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models Disharee Nath1,2, Julie R. White3,4, Gennady Bratslavsky1, Leszek Kotula1,2 1Department of Urology, SUNY Upstate Medical University, 2Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 3Laboratory of Comparative Pathology, Memorial Sloan-Kettering Cancer Center, 4Boulder BioPATH, Inc Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses. Immunology and Infection Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist Elisa Sanchez1,2, Morgan Huse1 1Immunology Program, Memorial Sloan-Kettering Cancer Center, 2Weill-Cornell Graduate School of Medical Sciences This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation. Cancer Research An In Vivo Murine Sciatic Nerve Model of Perineural Invasion Sylvie Deborde*1, Yasong Yu*1, Andrea Marcadis1, Chun-Hao Chen1, Ning Fan2, Richard L. Bakst3, Richard J. Wong1 1Department of Surgery, Memorial Sloan Kettering Cancer Center, 2Molecular Cytology Core Facility, Memorial Sloan Kettering Cancer Center, 3Department of Radiation Oncology, Mount Sinai Hospital We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion. Genetics Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma Jonathan B. Reichel1, Jason McCormick2, Jonathan R. Fromm3, Olivier Elemento4, Ethel Cesarman5, Mikhail Roshal6 1Innovation Laboratory, Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, 2Flow-Sorting Core Facility, Weill Cornell Medical College, 3Department of Laboratory Medicine, University of Washington, 4Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Cornell Medical College, 5Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, 6Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Immunology and Infection Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus Shan F. Brunel1, Jude M. Bain1, Jill King1, Lena J. Heung2, Shinji Kasahara2, Tobias M. Hohl2, Adilia Warris1 1Aberdeen Fungal Group, MRC Centre for Medical Mycology, Institute of Medical Sciences, University of Aberdeen, 2Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, US Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth. Bioengineering A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting Gabriel E. Büchel1,2, Brandon Carney1,3, Jun Tang1, Brian M. Zeglis1,3, Jörg Eppinger2, Thomas Reiner1,4 1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2KAUST Catalysis Center, King Abdullah University of Science and Technology, 3Department of Chemistry, Hunter College, and PhD Program in Chemistry, Graduate Center of City University of New York, 4Department of Radiology, Weill Cornell Medical College This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed. Genetics Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development Zhong-Dong Shi*1, Chew-Li Soh*1, Zengrong Zhu*1, Danwei Huangfu1 1Developmental Biology Program, Sloan Kettering Institute Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive β-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression. Cancer Research A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines Jun Tang1, Carlos Pérez-Medina1,2, Yiming Zhao2, Ahmad Sadique1, Willem J. M. Mulder2, Thomas Reiner1 1Department of Radiology, Memorial Sloan Kettering Cancer Center, 2Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation. Immunology and Infection Rapid Molecular Detection and Differentiation of Influenza Viruses A and B Caitlin C. Otto1, Samuel E. Kaplan1, Jeffrey Stiles1, Albina Mikhlina1, Cindy Lee1, N. Esther Babady1, Yi-Wei Tang1 1Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform. Medicine Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis Surya Kumari Vadrevu1, Sharad Sharma1,2, Navin Chintala1,3, Jalpa Patel1, Magdalena Karbowniczek1, Maciej Markiewski1 1Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Science Center, 2Merck Research Labs, 3Department of Surgery, Memorial Sloan Kettering Cancer Center Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used. Developmental Biology Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells Scott J. Callahan1,2, Yvonne Mica3, Lorenz Studer1 1The Center for Stem Cell Biology, Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, 2Cancer Biology and Genetics Program, Gerstner Sloan-Kettering Graduate School, Sloan-Kettering Institute for Cancer Research, 3Thermo Fisher Scientific This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol. Medicine Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells Estrelania S. Williams1, Veronica Rodriguez-Bravo3, Uma Chippada-Venkata2, Janis De Ia Iglesia-Vicente1, Yixuan Gong2, Matthew Galsky2, William Oh2, Carlos Cordon-Cardo1, Josep Domingo-Domenech1 1Department of Pathology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 2Department of Hematology/Oncology, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, 3Molecular Biology Program, Memorial Sloan-Kettering Cancer Center This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men. Biology TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos Benjamin Bertin1, Yoan Renaud1, Rajaguru Aradhya2, Krzysztof Jagla1, Guillaume Junion1 1University of Clermont-Ferrand, 2Memorial Sloan Kettering Cancer Center Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos. Chemistry The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies Brian M. Zeglis1, Jason S. Lewis1 1Department of Radiology, Memorial Sloan Kettering Cancer Center Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described. Bioengineering Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging Thomas Reiner*1, Jason S. Lewis1, Brian M. Zeglis*1 1Department of Radiology, Memorial Sloan Kettering Cancer Center The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand. Medicine Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics Adriana D. Corben*1, Mohammad M. Uddin*2, Brooke Crawford3, Mohammad Farooq4, Shanu Modi5, John Gerecitano5, Gabriela Chiosis2, Mary L. Alpaugh6 1Department of Pathology, Memorial Sloan Kettering Cancer Center, 2Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, 3Department of Radiology, Weill Cornell Medical College, 4Department of Medicine, Memorial Sloan Kettering Cancer Center, 5Department of Oncology, Memorial Sloan Kettering Cancer Center, 6Department of Surgery, Memorial Sloan Kettering Cancer Center Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes. Medicine Femoral Bone Marrow Aspiration in Live Mice Young Rock Chung1, Eunhee Kim1, Omar Abdel-Wahab1 1Human Oncology and Pathogenesis Program, Department of Medicine, Memorial Sloan-Kettering Cancer Center This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies. Medicine Isolation of Cancer Stem Cells From Human Prostate Cancer Samples Samuel J. Vidal1, S. Aidan Quinn1, Janis de la Iglesia-Vicente1, Dennis M. Bonal1, Veronica Rodriguez-Bravo2, Adolfo Firpo-Betancourt1, Carlos Cordon-Cardo1, Josep Domingo-Domenech1 1Department of Pathology, Icahn School of Medicine at Mount Sinai, 2Molecular Biology Program, Memorial Sloan-Kettering Cancer Center The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps. Biology Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing Helen H Won1, Sasinya N Scott1, A. Rose Brannon1, Ronak H Shah1, Michael F Berger1,2 1Department of Pathology, Memorial Sloan-Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations. Biology Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice Yu Chen1,2, Tambudzai Shamu2, Hui Chen3, Peter Besmer3, Charles L. Sawyers2,4, Ping Chi1,5 1Department of Medicine, Memorial Sloan Kettering Cancer Center, 2Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, 3Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 4Howard Hughes, Medical Institute, 5Laboratory of Chromatin Biology and Epigenetics, The Rockefeller University The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.
