Dana-Farber Cancer Institute View Institution's Website 16 articles published in JoVE Cancer Research ATAC-Seq Optimization for Cancer Epigenetics Research Mikhala Cooper*1, Atrayee Ray*1, Atrayee Bhattacharya2, Archana Dhasarathy1, Motoki Takaku1 1University of North Dakota School of Medicine and Health Sciences, 2Dana-Farber Cancer Institute ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells. Biology A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury Hussein Atta*1,2, Ali Hashemi Gheinani*1,2, Amanda Wacker1, Yaser Heshmati3,4,5, Alex Bigger-Allen1,6, George Lambrinos1,2, Yao Gao2,7, Diane R. Bielenberg2,7, Rosalyn M. Adam1,2 1 The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry. Cancer Research Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids Erin R. Bonner1,2, Karim Saoud1, Sulgi Lee1,2, Eshini Panditharatna3, Madhuri Kambhampati1, Sabine Mueller4,5, Javad Nazarian1,2,5 1 Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response. Biochemistry Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells Shellaina J.V. Gordon*1, Yao Xiao*2, Amanda L. Paskavitz3, Napoleón Navarro-Tito4, Juan G. Navea2, Teresita Padilla-Benavides1 1Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 2Department of Chemistry, Skidmore College, 3Candiac MR Center, Beth Israel Deaconess Medical Center, 4Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Guerrero Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy. 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. Developmental Biology A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening Charles A. C. Williams1, Nathanael S. Gray2,3, Greg M. Findlay1 1The MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, 2Department of Cancer Biology, Dana-Farber Cancer Institute, 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the naïve-primed pluripotent transition. Developmental Biology Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae Elliott J. Hagedorn1,2, Jennifer L. Cillis3, Caitlyn R. Curley3, Taylor C. Patch3, Brian Li1,2, Bradley W. Blaser1,2,7, Raquel Riquelme1,2, Leonard I. Zon1,2,4,5,6, Dhvanit I. Shah1,2,3,4,5 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest. Biology Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites Erik Serrao1, Peter Cherepanov2, Alan N. Engelman1 1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 2Chromatin Structure and Mobile DNA, The Francis Crick Institute We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools. Medicine A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies Emily J. Chadwick1, David P. Yang1, Mariella G. Filbin2, Emanuele Mazzola3, Yu Sun1, Oded Behar1,4, Maria F. Pazyra-Murphy1, Liliana Goumnerova5, Keith L. Ligon6, Charles D. Stiles1, Rosalind A. Segal1 1Department of Cancer Biology, Dana-Farber Cancer Institute, 2 Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. Biology Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 Daniel E. Bauer*1,2,3, Matthew C. Canver*1, Stuart H. Orkin1,2,3,4 1Harvard Medical School, 2 CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9. Immunology and Infection A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions Mandy Muller*1,2, Patricia Cassonnet*1, Michel Favre1, Yves Jacob1,3, Caroline Demeret1 1Unité de Génétique, Papillomavirus et Cancer Humain (GPCH), Institut Pasteur, 2Cellule Pasteur, Université Sorbonne Paris Cité, 3Center for Cancer Systems Biology (CCSB), Harvard Medical School, Department of Cancer Biology, Dana Farber Cancer Institute This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA. Biology Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells Susan Fotheringham1,2, Keren Levanon1,2,3, Ronny Drapkin1,2,4 1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis. Immunology and Infection Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade Jeff K. Davies1,2, Christine M. Barbon1, Annie R. Voskertchian1, Lee M. Nadler1,2, Eva C. Guinan3,4 1Medical Oncology, Dana Farber Cancer Institute, 2Department of Medicine, Brigham and Womens Hospital, 3Pediatric Oncology, Dana Farber Cancer Institute, 4Division of Hematology/Oncology, Children’s Hospital Boston This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation. Biology Retro-orbital Injection in Adult Zebrafish Emily K. Pugach1,2, Pulin Li1,2, Richard White1,2,3, Leonard Zon1,2 1Department of Hematology and Oncology, Children’s Hospital Boston, 2Harvard Stem Cell Institute, Harvard Medical School, 3Department of Medical Oncology, Dana Farber Cancer Institute Here we show how to do retro-orbital injection in adult zebrafish. Biology Olfactory Behavioral Testing in the Adult Mouse Rochelle M. Witt1,2, Meghan M. Galligan1,2, Jennifer R. Despinoy1,2, Rosalind Segal1,2 1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School Fundamental, yet unique properties of the rodent olfactory system have led to its increasing study among biologists. A relatively simple assessment of its function is then also needed. Here we describe sensitive tests for the characterization of mouse olfactory sensitivity and preference. Biology Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures Maria F. Pazyra-Murphy1,2, Rosalind A. Segal1,2 1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.