Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

Summary

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

Abstract

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3⁺), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3⁺), and T helper 17 (Th17) cells (i.e., IL-17⁺CD3⁺) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17⁺ non-T cells. This suggests that IL-17⁺ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Introduction

Oropharyngeal squamous cell carcinomas (OPSCCs) are a heterogeneous group of squamous cell cancers originating in the oropharynx. Risk factors for OPSCC include human papillomavirus (HPV) infection and alcohol and tobacco use^1,2. The role of the immune response and how to use this in a clinical setting is just starting to be explored. The tumor microenvironment is infiltrated by immune cells that are attracted to the cancer site. Although a high CD8⁺ cytotoxic T-cell frequency has been correlated with improved survival in OPSCC patients³, the role of other T-cell subsets, inc....

Protocol

Patient samples were handled according to the medical ethical guidelines described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies (www.federa.org).

1. Prepare Slides

1. Obtain resected tissue material (any kind of tissue) from a pathology department after obtaining medical ethical committee permission to use resected material.
   NOTE: For the current experiment, pretreatment tumor samples were obtained from primary oropharyngeal tumors diagnosed in the Leiden University Medical Center, Leiden, the Netherlands between 1970 and 2011, selected as describe....

Results

A series of FFPE pretreatment tumor samples obtained from primary oropharyngeal tumors diagnosed in the Leiden University Medical Center, Leiden, the Netherlands between 1970 and 2011, selected as described before (n = 162), was stained using the protocol described¹³. One to four random images of each slide were analyzed (Figure 1). Some autofluorescent cells are indicated, which can clearly be distinguished by their complete yellow ap.......

Discussion

For the protocol described, one of the most critical steps is to determine the correct dilution of the primary antibodies used. The dilution of the labeled secondary antibodies was 1:200, as recommended by the manufacturer of these specific Alexa-labeled antibodies. The dilution of the primary antibodies should then be determined by a serial dilution, preferably using two different samples of the intended tissue type (in this case, OPSCC). The optimal working dilution is the dilution at which a clear signal is obtained, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pathos Delta Ultra Rapid Tissue Processor	Milestone and Histostar		Automated tissue processor
Histostar	Thermo Scientific		Tissue block embedding machine
Formaldehyde	Baker
Xylol	Merck
Ethanol	Merck
milliQ water	Elaga Purelab Chorus
Paraffin wax/Paraclean	Klinipath	5079A
Microtome tissue holder	Leica	RM225
Flex IHC side	Dako		Tissue slide
Tris	Merk-Milipore	1,083,821,000
EDTA	Baker	1073
PBS	Bio-Rad	BUF036A
BSA	Sigma	A9647
Rabbit anti-CD3	Abcam	ab828	Titrate required antibody dilution
Mouse IgG1 anti-FoxP3	Abcam	ab20034	Titrate required antibody dilution
Goat IgG anti-IL-17	R&D Systems	AF-317-NA	Titrate required antibody dilution
Rabbit Ig isotype control antibody	Abcam	ab27472	Use at same final concentration as anti-CD3
Mouse IgG1 isotype control antibody	Abcam	ab91353	Use at same final concentration as anti-FoxP3
Goat IgG isotype control antibody	ThermoFisher Scientific	02-6202	Use at same final concentration as anti-IL-17
Donkey anti-rabbit IgG A546	ThermoFisher Scientific	A10040	Dilute 1:200 in 1% BSA/PBS
Donkey anti-mouse-A647	ThermoFisher Scientific	A31571	Dilute 1:200 in 1% BSA/PBS
Donkey anti-goat IgG A488	ThermoFisher Scientific	A11055	Dilute 1:200 in 1% BSA/PBS
VectaShield containing DAPI	Vector Laboratories	H-1200
LSM700 confocal laser scanning microscope	Zeiss
LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective	Zeiss	420852-9972-720
LSM Zen Software	Zeiss	version 2009
LSM Image Browser	Zeiss	version 4.2.0.121	Available to download at www.zeiss.com/microscopy/int/website/downloads/lsm-image-browser.html.
SPSS	IBM Corp.	version 20.0
ImageJ		version 1.50i	Available to download at http://rsb.info.nih.gov/ij.