Memorial Sloan Kettering Cancer Center

26 articles published in JoVE

• Biochemistry

  
  An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics Anthony Gaba*¹, Hongyun Wang*¹, Xiaohui Qu^{1 1}Molecular 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 Allon^2,4, Moshe Elkabets^{1,2 1}The 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. Sawyers^1,2, Wouter R. Karthaus^{1 1}Human 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 Berisha^1,2, Suchetan Pal¹, Moritz F. Kircher^{1,3,4,5,6,7 1}Department 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 Roi^1,2, Pierre Adumeau¹, Brian M. Zeglis^{1,2,3,4 1}Department 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 Membreno^1,2, Brendon E. Cook^1,2,3, Brian M. Zeglis^{1,2,3,4 1}Department 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 Nath^1,2, Julie R. White^3,4, Gennady Bratslavsky¹, Leszek Kotula^{1,2 1}Department 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 Sanchez^1,2, Morgan Huse^{1 1}Immunology 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. Wong^{1 1}Department 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 Roshal^{6 1}Innovation 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 Warris^{1 1}Aberdeen 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. Büchel^1,2, Brandon Carney^1,3, Jun Tang¹, Brian M. Zeglis^1,3, Jörg Eppinger², Thomas Reiner^{1,4 1}Department 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 Huangfu^{1 1}Developmental 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-Medina^1,2, Yiming Zhao², Ahmad Sadique¹, Willem J. M. Mulder², Thomas Reiner^{1 1}Department 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 Tang^{1 1}Department 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 Sharma^1,2, Navin Chintala^1,3, Jalpa Patel¹, Magdalena Karbowniczek¹, Maciej Markiewski^{1 1}Department 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. Callahan^1,2, Yvonne Mica³, Lorenz Studer^{1 1}The 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-Domenech^{1 1}Department 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 Junion^{1 1}University 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. Lewis^{1 1}Department 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 1}Department 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. Alpaugh^{6 1}Department 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-Wahab^{1 1}Human 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-Domenech^{1 1}Department 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 Berger^{1,2 1}Department 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 Chen^1,2, Tambudzai Shamu², Hui Chen³, Peter Besmer³, Charles L. Sawyers^2,4, Ping Chi^{1,5 1}Department 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.