Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes

  1. Jia-Ren Lin
  2. Benjamin Izar
  3. Shu Wang
  4. Clarence Yapp
  5. Shaolin Mei
  6. Parin M Shah
  7. Sandro Santagata
  8. Peter K Sorger  Is a corresponding author
  1. Harvard Medical School, United States
  2. Dana-Farber Cancer Institute, United States

Abstract

The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Intensity data used to generate figures is available in supplementary materials and can be downloaded from the HMS LINCS Center Publication Page (http://lincs.hms.harvard.edu/lin-elife-2018/) (RRID:SCR_016370). The images described are available at http://www.cycif.org/ (RRID:SCR_016267) and via and OMERO server as described at the LINCS Publication Page.

Article and author information

Author details

  1. Jia-Ren Lin

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  2. Benjamin Izar

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  3. Shu Wang

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  4. Clarence Yapp

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  5. Shaolin Mei

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  6. Parin M Shah

    Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States
    Competing interests
    No competing interests declared.
  7. Sandro Santagata

    Laboratory of Systems Pharmacology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  8. Peter K Sorger

    Department of Systems Biology, Harvard Medical School, Boston, United States
    For correspondence
    peter_sorger@hms.harvard.edu
    Competing interests
    Peter K Sorger, PKS is a member of the Board of Directors of RareCyte Inc., which manufactures the slide scanner used in this study, and co-founder of Glencoe Software, which contributes to and supports open-source OME/OMERO image informatics software. Other authors have no competing financial interests to disclose..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3364-1838

Funding

National Institutes of Health (P50GM107618)

  • Peter K Sorger

Dana-Farber/Harvard Cancer Center (GI SPORE Developmental Research Project Award)

  • Benjamin Izar

National Institutes of Health (U54HL127365)

  • Peter K Sorger

National Institutes of Health (R41-CA224503)

  • Peter K Sorger

Dana-Farber/Harvard Cancer Center (Claudia Adams Barr Program)

  • Benjamin Izar

National Institutes of Health (K08CA222663)

  • Benjamin Izar

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: Formalin fixed and paraffin embedded (FFPE) tissues from were retrieved from the archives of the Brigham and Women's Hospital as part of discarded/excess tissue protocols or obtained from commercial vendors. The Institutional Review Board (IRB) of the Harvard Faculty of Medicine last reviewed the research described in this paper on 2/16/2018 (under IRB17-1688) and judged it to 'involve no more than minimal risk to the subjects' and thus eligible for a waiver of the requirement to obtain consent as set out in 45CFR46.116(d). Tumor tissue and FFPE specimens were collected from patients under IRB-approved protocols (DFCI 11-104) at Dana-Farber Cancer Institute/Brigham and Women's Hospital, Boston, Massachusetts. The consent waiver described above also covers these tissues and specimens.

Reviewing Editor

  1. Arjun Raj, University of Pennsylvania, United States

Publication history

  1. Received: September 1, 2017
  2. Accepted: June 29, 2018
  3. Accepted Manuscript published: July 11, 2018 (version 1)
  4. Accepted Manuscript updated: July 12, 2018 (version 2)
  5. Version of Record published: August 3, 2018 (version 3)

Copyright

© 2018, Lin et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Jia-Ren Lin
  2. Benjamin Izar
  3. Shu Wang
  4. Clarence Yapp
  5. Shaolin Mei
  6. Parin M Shah
  7. Sandro Santagata
  8. Peter K Sorger
(2018)
Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes
eLife 7:e31657.
https://doi.org/10.7554/eLife.31657

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