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.

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, 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

  • 38,356
    views
  • 4,017
    downloads
  • 568
    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

    Changes in local mineral homeostasis facilitate the formation of benign and malignant testicular microcalcifications

    Ida Marie Boisen, Nadia Krarup Knudsen ... Martin Blomberg Jensen
    Research Article

    Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.

    1. Cancer Biology

    TAK1-mediated phosphorylation of PLCE1 represses PIP2 hydrolysis to impede esophageal squamous cancer metastasis

    Qianqian Ju, Wenjing Sheng ... Cheng Sun
    Research Article

    TAK1 is a serine/threonine protein kinase that is a key regulator in a wide variety of cellular processes. However, the functions and mechanisms involved in cancer metastasis are still not well understood. Here, we found that TAK1 knockdown promoted esophageal squamous cancer carcinoma (ESCC) migration and invasion, whereas TAK1 overexpression resulted in the opposite outcome. These in vitro findings were recapitulated in vivo in a xenograft metastatic mouse model. Mechanistically, co-immunoprecipitation and mass spectrometry demonstrated that TAK1 interacted with phospholipase C epsilon 1 (PLCE1) and phosphorylated PLCE1 at serine 1060 (S1060). Functional studies revealed that phosphorylation at S1060 in PLCE1 resulted in decreased enzyme activity, leading to the repression of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. As a result, the degradation products of PIP2 including diacylglycerol (DAG) and inositol IP3 were reduced, which thereby suppressed signal transduction in the axis of PKC/GSK-3β/β-Catenin. Consequently, expression of cancer metastasis-related genes was impeded by TAK1. Overall, our data indicate that TAK1 plays a negative role in ESCC metastasis, which depends on the TAK1-induced phosphorylation of PLCE1 at S1060.

    1. Cancer Biology
    2. Cell Biology

    Cell crowding activates pro-invasive mechanotransduction pathway in high-grade DCIS via TRPV4 inhibition and cell volume reduction

    Xiangning Bu, Nathanael Ashby ... Inhee Chung
    Research Article

    Cell crowding is a common microenvironmental factor influencing various disease processes, but its role in promoting cell invasiveness remains unclear. This study investigates the biomechanical changes induced by cell crowding, focusing on pro-invasive cell volume reduction in ductal carcinoma in situ (DCIS). Crowding specifically enhanced invasiveness in high-grade DCIS cells through significant volume reduction compared to hyperplasia-mimicking or normal cells. Mass spectrometry revealed that crowding selectively relocated ion channels, including TRPV4, to the plasma membrane in high-grade DCIS cells. TRPV4 inhibition triggered by crowding decreased intracellular calcium levels, reduced cell volume, and increased invasion and motility. During this process, TRPV4 membrane relocation primed the channel for later activation, compensating for calcium loss. Analyses of patient-derived breast cancer tissues confirmed that plasma membrane-associated TRPV4 is specific to high-grade DCIS and indicates the presence of a pro-invasive cell volume reduction mechanotransduction pathway. Hyperosmotic conditions and pharmacologic TRPV4 inhibition mimicked crowding-induced effects, while TRPV4 activation reversed them. Silencing TRPV4 diminished mechanotransduction in high-grade DCIS cells, reducing calcium depletion, volume reduction, and motility. This study uncovers a novel pro-invasive mechanotransduction pathway driven by cell crowding and identifies TRPV4 as a potential biomarker for predicting invasion risk in DCIS patients.