Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue

Abstract Multiplex immunofluorescence (mIF) has arisen as an important tool for immuno-profiling tumor tissues. We updated our manual protocol into an automated protocol that allows the use of up to seven markers in five mIF panels to apply to formalin-fixed paraffin-embedded tumor tissues. Using a...

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Autores principales:	Edwin Roger Parra, Maria C. Ferrufino-Schmidt, Auriole Tamegnon, Jiexin Zhang, Luisa Solis, Mei Jiang, Heladio Ibarguen, Cara Haymaker, J. Jack Lee, Chantale Bernatchez, Ignacio Ivan Wistuba
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/efe9023c4040440db307e28f3a0467e4
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spelling	oai:doaj.org-article:efe9023c4040440db307e28f3a0467e42021-12-02T13:44:14ZImmuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue10.1038/s41598-021-88156-02045-2322https://doaj.org/article/efe9023c4040440db307e28f3a0467e42021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-88156-0https://doaj.org/toc/2045-2322Abstract Multiplex immunofluorescence (mIF) has arisen as an important tool for immuno-profiling tumor tissues. We updated our manual protocol into an automated protocol that allows the use of up to seven markers in five mIF panels to apply to formalin-fixed paraffin-embedded tumor tissues. Using a tyramide signal amplification system, we optimized five mIF panels that included cytokeratin to characterize malignant cells (MCs), immune checkpoint markers (i.e., PD-L1, B7-H3, B7-H4, IDO-1, VISTA, LAG3, ICOS, TIM3, and OX40), tumor-infiltrating lymphocytic markers (i.e., CD3, CD8, CD45RO, granzyme B, PD-1, and FOXP3), and markers to characterize myeloid-derived suppressor cells (i.e., CD68, CD66b, CD14, CD33, Arg-1, and CD11b). To determine analytical reproducibility and the impact of those panels for immuno-profiling tumor tissues, we performed an exploratory analysis in a set of non–small cell lung cancer (NSCLC) samples. The slides were scanned, and the different cell phenotypes were quantified by simultaneous co-localizations with the markers using image analysis software. Comparison between the time points of staining showed high analytical reproducibility. The analysis of NSCLC cases showed an immunosuppressive microenvironment with PD-L1/PD-1 expression as a predominant axis. Interestingly, high density of MCs expressing B7-H4 was correlated with recurrence. Unexpectedly, MCs expressing OX40 were also detected, and those cells were a closer distance to CD3+T-cells than were MCs expressing other immune checkpoints. Two different cellular patterns of spatial distribution were determined according the CD3 distribution, and the predominant pattern was related with active immunosuppressive interaction with MCs. Our study shows that these five mIF panels can identify multiple targets in a single cell with high reproducibility. The study of different cell populations and their spatial relationship can open new ideas for therapeutic approaches.Edwin Roger ParraMaria C. Ferrufino-SchmidtAuriole TamegnonJiexin ZhangLuisa SolisMei JiangHeladio IbarguenCara HaymakerJ. Jack LeeChantale BernatchezIgnacio Ivan WistubaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Edwin Roger Parra Maria C. Ferrufino-Schmidt Auriole Tamegnon Jiexin Zhang Luisa Solis Mei Jiang Heladio Ibarguen Cara Haymaker J. Jack Lee Chantale Bernatchez Ignacio Ivan Wistuba Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
description	Abstract Multiplex immunofluorescence (mIF) has arisen as an important tool for immuno-profiling tumor tissues. We updated our manual protocol into an automated protocol that allows the use of up to seven markers in five mIF panels to apply to formalin-fixed paraffin-embedded tumor tissues. Using a tyramide signal amplification system, we optimized five mIF panels that included cytokeratin to characterize malignant cells (MCs), immune checkpoint markers (i.e., PD-L1, B7-H3, B7-H4, IDO-1, VISTA, LAG3, ICOS, TIM3, and OX40), tumor-infiltrating lymphocytic markers (i.e., CD3, CD8, CD45RO, granzyme B, PD-1, and FOXP3), and markers to characterize myeloid-derived suppressor cells (i.e., CD68, CD66b, CD14, CD33, Arg-1, and CD11b). To determine analytical reproducibility and the impact of those panels for immuno-profiling tumor tissues, we performed an exploratory analysis in a set of non–small cell lung cancer (NSCLC) samples. The slides were scanned, and the different cell phenotypes were quantified by simultaneous co-localizations with the markers using image analysis software. Comparison between the time points of staining showed high analytical reproducibility. The analysis of NSCLC cases showed an immunosuppressive microenvironment with PD-L1/PD-1 expression as a predominant axis. Interestingly, high density of MCs expressing B7-H4 was correlated with recurrence. Unexpectedly, MCs expressing OX40 were also detected, and those cells were a closer distance to CD3+T-cells than were MCs expressing other immune checkpoints. Two different cellular patterns of spatial distribution were determined according the CD3 distribution, and the predominant pattern was related with active immunosuppressive interaction with MCs. Our study shows that these five mIF panels can identify multiple targets in a single cell with high reproducibility. The study of different cell populations and their spatial relationship can open new ideas for therapeutic approaches.
format	article
author	Edwin Roger Parra Maria C. Ferrufino-Schmidt Auriole Tamegnon Jiexin Zhang Luisa Solis Mei Jiang Heladio Ibarguen Cara Haymaker J. Jack Lee Chantale Bernatchez Ignacio Ivan Wistuba
author_facet	Edwin Roger Parra Maria C. Ferrufino-Schmidt Auriole Tamegnon Jiexin Zhang Luisa Solis Mei Jiang Heladio Ibarguen Cara Haymaker J. Jack Lee Chantale Bernatchez Ignacio Ivan Wistuba
author_sort	Edwin Roger Parra
title	Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
title_short	Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
title_full	Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
title_fullStr	Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
title_full_unstemmed	Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
title_sort	immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/efe9023c4040440db307e28f3a0467e4
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Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue

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