Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues

Abstract

Immune-profiling is becoming an important tool to identify predictive markers for the response to immunotherapy. Our goal was to validate multiplex immunofluorescence (mIF) panels to apply to formalin-fixed and paraffin-embedded tissues using a set of immune marker antibodies, with the Opal™ 7 color Kit (PerkinElmer) in the same tissue section. We validated and we described two panels aiming to characterize the expression of PD-L1, PD-1, and subsets of tumor associated immune cells. Panel 1 included pancytokeratin (AE1/AE3), PD-L1, CD4, CD8, CD3, CD68, and DAPI, and Panel 2 included pancytokeratin, PD-1, CD45RO, granzyme B, CD57, FOXP3, and DAPI. After all primary antibodies were tested in positive and negative controls by immunohistochemistry and uniplex IF, panels were developed and simultaneous marker expressions were quantified using the Vectra 3.0™ multispectral microscopy and image analysis InForm™ 2.2.1 software (PerkinElmer).These two mIF panels demonstrated specific co-localization in different cells that can identify the expression of PD-L1 in malignant cells and macrophages, and different T-cell subpopulations. This mIF methodology can be an invaluable tool for tumor tissue immune-profiling to allow multiple targets in the same tissue section and we provide that is accurate and reproducible method when is performed carefully under pathologist supervision.

Breast cancer intratumour heterogeneity: current status and clinical implications

“…These multiplex immunostaining techniques are likely to improve our understanding of intratumour heterogeneity while maintaining the morphology of the lesions. For instance, multiplex immunofluorescence marker expression has recently been reported to be an important tool for tumour tissue immune profiling; it allowed the assay of multiple biomarker targets in the same tissue section, and can be used for the development of novel predictive biomarkers for cancer immunotherapy …”

In situ multiplex immunofluorescence analysis of the inflammatory burden in kidney allograft rejection: A new tool to characterize the alloimmune response

“…In particular, a novel detection strategy based on tyramide signal amplification (TSA), on paraffin‐embedded tissue section, is now available . Until now, this new technology has mainly been used in oncology to better characterize the in situ immune cell infiltrate in tumors …”

Novel Platforms of Multiplexed Immunofluorescence for Study of Paraffin Tumor Tissues

“…It is an enzymelinked signal amplification method that conventional is using to detect and localize low copy number of proteins present in tissue by IHC, using most commonly alkaline phosphatase or horseradish peroxidase (HRP) to catalyse the deposition of labelled tyramide molecules at the site of probe or epitope detection. Tyramides can be conjugated to biotin or fluorescent labels and labeled with streptavidin enzyme HRP 6,18 . The HRP catalyzes the formation of tyramide into highly reactive tyramide radicals that covalently bind to electron-rich tyrosine moieties on FFPE tissue.…”

“…The HRP catalyzes the formation of tyramide into highly reactive tyramide radicals that covalently bind to electron-rich tyrosine moieties on FFPE tissue. Tissue surfaces with anchored biotinylated tyramide, must be further treated with fluorescent or enzyme tagged proteins that have a high affinity for biotin as streptavidin before to microscopic visualization 6,18 . The detection of the proteins is increased more than 10-times compared to standard biotin-based staining methods 19 .…”

“…The protocol allows researchers to use antibodies raised in the same species, and different panels (Figure 3) combined different targets can be created using this technology 4,18 . The approach, in the manual protocol, involves detection with fluorescent TSA reagents, followed by microwave treatment that removes any nonspecific staining and reduces tissue autofluorescence for each antibody cycle.…”