Eight-Color Multiplex Immunohistochemistry for Simultaneous Detection of Multiple Immune Checkpoint Molecules within the Tumor Microenvironment

J Immunol. 2018 Jan 1;200(1):347-354. doi: 10.4049/jimmunol.1701262. Epub 2017 Nov 15.

Abstract

Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / therapeutic use*
  • Antigens, Neoplasm / immunology
  • Antigens, Neoplasm / metabolism
  • B7-H1 Antigen / immunology
  • B7-H1 Antigen / metabolism
  • Biomarkers, Tumor / metabolism
  • CTLA-4 Antigen / immunology
  • CTLA-4 Antigen / metabolism
  • Hepatitis A Virus Cellular Receptor 2 / immunology
  • Hepatitis A Virus Cellular Receptor 2 / metabolism
  • Humans
  • Immunohistochemistry / methods*
  • Immunotherapy / methods*
  • Neoplasms / diagnosis*
  • Neoplasms / immunology
  • Neoplasms / therapy
  • Programmed Cell Death 1 Receptor / immunology
  • Programmed Cell Death 1 Receptor / metabolism
  • Receptors, OX40 / immunology
  • Receptors, OX40 / metabolism
  • Single-Cell Analysis
  • Tumor Microenvironment*
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / immunology
  • Tumor Necrosis Factor Receptor Superfamily, Member 7 / metabolism

Substances

  • Antibodies, Monoclonal
  • Antigens, Neoplasm
  • B7-H1 Antigen
  • Biomarkers, Tumor
  • CTLA-4 Antigen
  • HAVCR2 protein, human
  • Hepatitis A Virus Cellular Receptor 2
  • PDCD1 protein, human
  • Programmed Cell Death 1 Receptor
  • Receptors, OX40
  • TNFRSF4 protein, human
  • Tumor Necrosis Factor Receptor Superfamily, Member 7