Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes
- Harvard Medical School, United States
- Dana-Farber Cancer Institute, United States
- Whitehead Institute for Biomedical Research, 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 analysed 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 website (http://lincs.hms.harvard.edu/lin-elife-2018/). The images described will be available at http://www.cycif.org/ (RRID:SCR_016267). and will be deposited at the EBI Image Data Repository (https://idr.openmicroscopy.org/).
Article and author information
Author details
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.
Copyright
© 2018, Sorger 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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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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