Bringing the Tumor Microenvironment into Focus: 

Simplified Development of Seven-Color Multiplex Immunohistochemistry-Immunofluorescence (mIF) Panels

With the advancement of immunotherapeutics, the need to understand the tumor microenvironment has never been more pressing. Recent advances in mIF and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers.

Melissa Whiteman, PhD, Eric McIntush, PhD, Michael Spencer, PhD

Abstract
With the advancement of immunotherapeutics, the need to understand the tumor microenvironment has never been more pressing. Multiplex immunofluorescence (mIF) maximizes the amount of data acquired from an individual sample. Recent advances in mIF and multispectral imaging facilitate accurate simultaneous analysis of multiple tissue markers. This is critical in instances where sample is limited, such as a tumor biopsy or other clinical specimen. The applications of mIF are numerous, and span clinical, translational, and basic research applications. A seven-color mIF can take eight weeks, or more, to develop. Herein, we describe a simplified, faster approach to seven-color mIF development.
FFPE human tissue was stained with PathPlex™ Panel 4 IHC validated primary antibodies (Bethyl Laboratories [A810-004]), mouse or rabbit HRP-conjugated secondary antibodies (Bethyl Laboratories [A90-116P, A120-501P]) and detected using Opal™ Polaris 7-color IHC kit fluorophores (Perkin Elmer [NEL861001KT]). Primary antibody order was optimized utilizing tissue microarray serial sections, and three slides per target by staining after the first, third, or sixth heat-induced epitope retrieval (HIER). All three slides were imaged using the same exposure time and analyzed for target/nucleus counts, signal intensity, and background. Finally, the order was tested in the seven-color mIF and compared to single stain for confirmation. Whole slide scans were generated using the Vectra Polaris® and analyzed using InForm® image analysis package.

Development time of a seven-color mIF was reduced using IHC validated antibodies and the optimized DAB dilution. Antibody order was guided by results of three slides stained after first, third or sixth HEIR. The ratio of target staining/DAPI nuclear counts, average intensity and overall background reveals the optimal order of staining. Some targets reveal optimal staining with greater intensity and lower background when stained last, such as FOXP3 (Step 2, Table 1 and Figure 1). For other antibodies, the opposite may be true or there could be no effect from multiple HIER. There are 720 possible combinations for a seven-color panel. Using this method, the number of slides was reduced to three per target (18) plus confirmation seven-color slides resulting in a panel containing CD3, CD8, CD68, Cytokeratin, FOXP3 and PD-L1 (Step 5, Figure 2).

Multiplex IF is a powerful technique that allows for examination of spatial arrangement of proteins of interest as well as protein interaction/co-localization of multiple targets within a single tissue specimen. mIF panels can take eight or more weeks to optimize, however, researchers can save time and resources using validated antibodies and this antibody order guide.

The information on this page was originally presented in poster form at SITC 2019. We have made the original poster available for download as a PDF for your convenience.
Introduction to Multiplex
R
Detect multiple antigens on the same tissue while conserving the sample
R

Provides colocalization and spatial orientation of proteins, which facilitates an accurate determination of the target’s subcellular localization, identification of multiple cell types, and resolution of the relative proximity of biomarkers

R

Obtain information on the expression levels of biomarkers while increasing the number of
biomarkers that can be visualized simultaneously on a single slide

R

Capture superior information about the tissue microenvironment

The information on this page was originally presented in poster form at SITC 2019. We have made the original poster available for download as a PDF for your convenience.
Basic Protocol
E

mIF is performed on formalin-fixed paraffin embedded tissue sections

E
A series of staining followed by heat treatment will bind fluorescent signal to the target
E
There is no requirement to use antibodies from different hosts (any variation of mouse and/or rabbit can be used)
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How Does It Work?
E

TSA multiplexing allows the user to choose any rabbit or mouse antibody without cross-reactivity: Select the best antibodies!

E
After labelling, the antibodies (primary and secondary) are removed or stripped by the heat-induction (heat-induced epitope retrieval) step
E
Substrate is covalently bonded to the target protein site when tyramide forms bonds with tyrosine residues on or near the antigen and the fluorophore is permanently deposited at the site of the antigen
E
Multiple rounds of staining are allowed by this process of stripping primary/secondary antibody pairs while preserving antigen-associate fluorescent signal
The information on this page was originally presented in poster form at SITC 2019. We have made the original poster available for download as a PDF for your convenience.
5 Steps to Creating a Custom Panel:

1

Select targets of interest and highest quality IHC validated antibodies

2

Determine panel order using serial tissue sections and stain slides after first, third and sixth HIER

3

Compare images to determine optimal order placement

4

Pair antibody with fluorophore

5

Confirmation mIF staining

Step 1

Select six antibodies of interest using Bethyl PathPlex™ Validated Antibodies

Select high quality IHC validated antibodies and confirm dilution needed for Opal™ reagents using single plex stains. Bethyl PathPlex™ antibodies are validated to work with Opal™ reagents and need no or minimal adjustments from the suggested optimal dilution. For select PathPlex™ antibodies the general staining order has been determined to guide the user during multiplex panel optimization.

Step 2

Using three slides per target, stain slides after first, third or sixth HIER to determine best signal

FOXP3 Staining After First, Third or Sixth HIER

Step 3

Ordering the antibodies for a Six Plex Panel

Step 4

Assign Fluorophore to Antibodies

Step 5

Order confirmation multiplex IHC

The information on this page was originally presented in poster form at SITC 2019. We have made the original poster available for download as a PDF for your convenience.
Bethyl PathPlex™ Panels
Panel
Catalog Number
Antibody
PathPlex™ Panel 1
CD3 CD8 PD-L1
PathPlex™ Panel 2
CD3 CD8 CD20
PathPlex™ Panel 3
CD3 CD20 CD68
PathPlex™ Panel 4
CD3 CD8 CD68 Cytoleratin FOXP3 PD-L1
PathPlex™ Panel 5
CD3 CD8 CD68 Cytoleratin Ki-67 PD-L1
PathPlex™ Panel 6
CD3 CD8 GranzymeB Cytokeratin Ki-67 Sox10
PathPlex™ Panel 7
CD4 CD8 CD45RO Cytokeratin FOXP3
PathPlex™ Panel 8
CD3 CD4 CD8 Cytokeratin FOXP3 LAG3
Or easily design your own custom panel using PathPlex™ Validated Antibodies
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Summary
Bethyl has introduced a series of PathPlex™ validated antibodies ready for use in mIF IHC. These antibodies have been validated for use with Opal™ fluorophores and pre-optimized order of staining using tonsil arrays. Order was confirmed on both tonsil and cancer array cores. PathPlex™ users should confirm staining using their preferred tissue since target abundance or absence can greatly affect results. Multiplex staining using all targets in the panel was simplified using the five steps to create a custom panel and the best signal table with PathPlex™ validated antibodies. This method reduces development time and resources and can be adapted for custom use.

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