MENU
JUN 20, 2019 1:30 PM PDT

Single Cell CNV End-to-End Workflow to Dissect Tumor Heterogeneity

C.E. Credits: P.A.C.E. CE Florida CE
Speaker
  • Postdoctoral Research Associate, Department of Translational Genomics, Keck School of Medicine, University of Southern California
    Biography
      Dr. Lee D. Gibbs is a postdoctoral research associate and USC Provost's scholar in the Department of Translational Genomics at Keck School of Medicine of University of Southern California under the mentorship of Dr. John D. Carpten. He works with a multi-disciplinary team to evaluate genomic alterations that are inherited and acquired during tumor development and to understand the clinical significance of these events. His research focuses on using novel genomic sequencing technologies to reveal the unexplored landscape of tumor heterogeneity spatially and at a single cell resolution. In addition, his research focuses on the discovery and validation of biomarkers and therapeutic targets in rare and aggressive cancers that affect minorities and medically underserved populations. Dr. Gibbs's goal is to evaluate the functional significance of these genomic events and their potential to determine prognosis, diagnosis, and therapeutic response.

    Abstract

    Tumor heterogeneity is a hallmark of cancer and can have significant impact on identifying drivers, including those that may be therapeutically relevant. Although, the traditional sequencing of bulk tumor specimens provides invaluable information, sequencing of individual cells provides a true representation of the cellular heterogeneity that exists within a cancer. Novel technologies such as single cell copy number variation (scCNV) enable copy number profiling at a single cell resolution. Single Cell CNV generates genome wide sequencing libraries from single cells to reveal tumor heterogeneity and evolution of subclonal populations. 

    In this study, we utilized a scCNV assay to assess heterogeneity within the 4T1 murine breast tumor model.  To accomplish this we have developed an end-to-end workflow including tissue dissociation, single cell library preparation, sequencing and data analysis. We injected 4T1 cells into the mammary fat pad of 4-6 week old BALB/c mice. Mice were sacrificed at 21 days and tumors were removed from the injection site and dissociated using Milteny’s Biotec Octo Dissociator. We utilized the 10X Genomics Chromium system to partition single cells and prepare sequencing libraries in parallel such that all DNA fragments produced within a partition are barcoded. Following library construction, the Illumina Novaseq 6000 system was used to sequence single cell libraries. Additionally, single cell libraries from a fraction of the 4T1 cells that were injected into the mammary fat and non-metastatic isogeneic cell line variants, 67NR and 168FARN, were also sequenced. 

    We observed a pair of amplifications (>8 copies) on chromosome 9 and three segments of amplification on chromosome 15 that were conserved throughout the injection site tumor cells. Interestingly, the amplifications on chromosome 15 encompass one of the most commonly observed breast cancer oncogenes, Myc, and the cell migration regulator, Skp2. Further, we observed three unique cluster of tumors cells which suggests the possibility of detecting major and minor clonal populations using this technology. 

    Our findings suggest scCNV will help us to improve characterizations of the entirety of a patient’s disease to uncover potential clones that are primed to induce metastasis, drug resistance, and relapse.

    Learning Objectives: 

    1. Apply single cell sequencing technologies to their ongoing projects that seek to develop precision therapies
    2. Identify subclonal populations at a single cell resolution
     


    Show Resources
    You May Also Like
    JAN 23, 2020 9:00 AM PST
    C.E. CREDITS
    JAN 23, 2020 9:00 AM PST
    DATE: January 23, 2020 TIME: 9:00am PST, 12:00pm EST...
    APR 07, 2020 8:00 AM PDT
    C.E. CREDITS
    APR 07, 2020 8:00 AM PDT
    DATE: April 7, 2020 TIME: 8:00am PT, 11:00am ET This webinar sets out to establish why quality control is key to robust, reliable, reproducible science. We will look at best practice criteri...
    MAY 08, 2020 10:00 AM PDT
    C.E. CREDITS
    MAY 08, 2020 10:00 AM PDT
    DATE: May 8, 2020 TIME: 10:00am PT, 11:00am MT, 1:00pm ET The application of next generation sequencing to interrogate immune repertoires and methods in which these highly complex dataset...
    FEB 26, 2020 9:00 AM PST
    C.E. CREDITS
    FEB 26, 2020 9:00 AM PST
    DATE: February 26, 2020 TIME: 9:00am PST 3D cell culture and analysis and the study of organoids and spheroids are becoming more prevalent as a research method in publications as traditional...
    FEB 19, 2020 11:00 AM PST
    C.E. CREDITS
    FEB 19, 2020 11:00 AM PST
    DATE: February 19, 2020TIME: 11:00am PST, 2:00pm EST...
    DEC 05, 2019 9:00 AM PST
    C.E. CREDITS
    DEC 05, 2019 9:00 AM PST
    DATE: December 5, 2019TIME: 9:00am PST, 12:00pm EST Characterization of the spatial distribution and abundance of proteins and mRNAs with morphological context within tissues e...
    Loading Comments...
    Show Resources