National Cancer Institute 12 articles published in JoVE Cancer Research Translational Orthotopic Models of Glioblastoma Multiforme Rajaa El Meskini1, Devon Atkinson1, Zoë Weaver Ohler1 1Center for Advanced Preclinical Research, Frederick National Laboratory for Cancer Research, National Cancer Institute Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology. Cancer Research Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells Meghan L. Good*1,2, Raul Vizcardo*1,2, Takuya Maeda1,2, Naritaka Tamaoki1,2, Parisa Malekzadeh1, Hiroshi Kawamoto3, Nicholas P. Restifo1,2 1Surgery Branch, National Cancer Institute, NIH, 2Center for Cell-Based Therapy, National Cancer Institute, NIH, 3Laboratory of Immunology, Institute for Frontier Life and Medical Sciences, Kyoto University This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8αβ+ single positive T cells using OP9/DLL1 co-culture system. Cancer Research A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants Raul Vizcardo*1,2, S.M. Rafiqul Islam*1,2, Takuya Maeda1,2, Naritaka Tamaoki1,2, Meghan L. Good1,2, Nicholas D. Klemen1,2, Marta Bosch-Marce1,2, Li Jia1, Mikhael J. Kruhlak3, Nicholas P. Restifo1,2 1Surgery Branch, National Cancer Institute, NIH, 2Center for Cell-Based Therapy, National Cancer Institute, NIH, 3Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, NIH This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to naïve T cells with the capacity for proliferation, memory formation, and tumor suppression. Immunology and Infection Merkel Cell Polyomavirus Infection and Detection Wei Liu*1, Nathan A. Krump*1, Christopher B. Buck2, Jianxin You1 1Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Laboratory of Cellular Oncology, National Cancer Institute Here, we present a protocol to infect primary human dermal fibroblast with MCPyV. The protocol includes isolation of dermal fibroblasts, preparation of MCPyV virions, virus infection, immunofluorescence staining, and fluorescence in situ hybridization. This protocol can be extended for characterizing MCPyV-host interactions and discovering other cell types infectable by MCPyV. Cancer Research Evaluating In Vitro DNA Damage Using Comet Assay Yanxin Lu1,2, Yang Liu1, Chunzhang Yang1 1Neuro-Oncology Branch, Center for Cancer Research, National Cancer Institute, 2Basic Medical Science Department, Zunyi Medical College-Zhuhai Campus The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy. Genetics Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards Joshua R. Porter1, William G. Telford2, Eric Batchelor1 1Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, 2Experimental Transplantation and Immunology Branch, Center for Cancer Research, National Cancer Institute We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts. Immunology and Infection Visualization of IL-22-expressing Lymphocytes Using Reporter Mice Wei Shen1, Wenqing Li1, Julie A. Hixon1, Caroline Andrews1, Scott K. Durum1 1Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse. Bioengineering Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique Kazuhide Sato1, Peter L. Choyke1, Kobayashi Hisataka1 1Molecular Imaging Program, National Cancer Institute Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT). Medicine In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells Carrie D. House1, Lidia Hernandez1, Christina M. Annunziata1 1Women's Malignancies Branch, National Cancer Institute Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells. Biology Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis Katie L. DeCicco-Skinner1, Gervaise H. Henry1, Christophe Cataisson2, Tracy Tabib1, J. Curtis Gwilliam1, Nicholas J. Watson1, Erica M. Bullwinkle1, Lauren Falkenburg1, Rebecca C. O'Neill1, Adam Morin1, Jonathan S. Wiest2 1Department of Biology, American University, 2Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified. Immunology and Infection Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors Steven J. Smith1, Stephen H. Hughes1 1HIV Drug Resistance Program, National Cancer Institute Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1. Biology Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis Lyuba Khavrutskii1,2, Joanna Yeh1, Olga Timofeeva3, Sergey G. Tarasov4, Samuel Pritt1, Karen Stefanisko1, Nadya Tarasova1 1Cancer and Inflammation Program, National Cancer Institute, 2Basic Science Program, SAIC-Frederick, Inc., 3Departments of Oncology and Radiation Medicine, Georgetown University Medical Center, 4Structural Biophysics Laboratory, National Cancer Institute Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.
