1. Cancer Biology
  2. Computational and Systems Biology

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
https://doi.org/10.7554/eLife.31657
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Reviewing Editor

  1. Arjun Raj, University of Pennsylvania, United States

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.

Version 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.

Metrics

  • 35,094
    views
  • 3,693
    downloads
  • 485
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Share this article

https://doi.org/10.7554/eLife.31657

Categories and tags

Research organism

Further reading

    1. Cancer Biology
    2. Cell Biology

    CYRI-B-mediated macropinocytosis drives metastasis via lysophosphatidic acid receptor uptake

    Savvas Nikolaou, Amelie Juin ... Laura M Machesky
    Research Article Updated

    Pancreatic ductal adenocarcinoma carries a dismal prognosis, with high rates of metastasis and few treatment options. Hyperactivation of KRAS in almost all tumours drives RAC1 activation, conferring enhanced migratory and proliferative capacity as well as macropinocytosis. Macropinocytosis is well understood as a nutrient scavenging mechanism, but little is known about its functions in trafficking of signalling receptors. We find that CYRI-B is highly expressed in pancreatic tumours in a mouse model of KRAS and p53-driven pancreatic cancer. Deletion of Cyrib (the gene encoding CYRI-B protein) accelerates tumourigenesis, leading to enhanced ERK and JNK-induced proliferation in precancerous lesions, indicating a potential role as a buffer of RAC1 hyperactivation in early stages. However, as disease progresses, loss of CYRI-B inhibits metastasis. CYRI-B depleted tumour cells show reduced chemotactic responses to lysophosphatidic acid, a major driver of tumour spread, due to impaired macropinocytic uptake of the lysophosphatidic acid receptor 1. Overall, we implicate CYRI-B as a mediator of growth and signalling in pancreatic cancer, providing new insights into pathways controlling metastasis.

    1. Cancer Biology

    FAK loss reduces BRAFV600E-induced ERK phosphorylation to promote intestinal stemness and cecal tumor formation

    Chenxi Gao, Huaibin Ge ... Jing Hu
    Research Article

    BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a ‘just-right’ level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAFLSL-V600E/+;Ptk2fl/fl mice, Fak deletion maximized BRAFV600E’s oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a ‘just-right’ ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.

    1. Cancer Biology
    2. Computational and Systems Biology

    Optimal transport for automatic alignment of untargeted metabolomic data

    Marie Breeur, George Stepaniants ... Vivian Viallon
    Research Article

    Untargeted metabolomic profiling through liquid chromatography-mass spectrometry (LC-MS) measures a vast array of metabolites within biospecimens, advancing drug development, disease diagnosis, and risk prediction. However, the low throughput of LC-MS poses a major challenge for biomarker discovery, annotation, and experimental comparison, necessitating the merging of multiple datasets. Current data pooling methods encounter practical limitations due to their vulnerability to data variations and hyperparameter dependence. Here, we introduce GromovMatcher, a flexible and user-friendly algorithm that automatically combines LC-MS datasets using optimal transport. By capitalizing on feature intensity correlation structures, GromovMatcher delivers superior alignment accuracy and robustness compared to existing approaches. This algorithm scales to thousands of features requiring minimal hyperparameter tuning. Manually curated datasets for validating alignment algorithms are limited in the field of untargeted metabolomics, and hence we develop a dataset split procedure to generate pairs of validation datasets to test the alignments produced by GromovMatcher and other methods. Applying our method to experimental patient studies of liver and pancreatic cancer, we discover shared metabolic features related to patient alcohol intake, demonstrating how GromovMatcher facilitates the search for biomarkers associated with lifestyle risk factors linked to several cancer types.