TY - JOUR
T1 - Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence
AU - Mori, Hidetoshi
AU - Bolen, Jennifer
AU - Schuetter, Louis
AU - Massion, Pierre
AU - Hoyt, Clifford C.
AU - VandenBerg, Scott
AU - Esserman, Laura
AU - Borowsky, Alexander D.
AU - Campbell, Michael J.
N1 - Funding Information:
This work was supported by NCI/NIH U01CA196406. We thank the MCL Pathology Working Group especially Angelo DeMarzo and Steve Dubinet for help in designing the panels, Wo Il Ju for editorial support and Wyatt Millstone for assistance with writing R scripts.
Publisher Copyright:
© 2021, The Author(s).
Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2020/12
Y1 - 2020/12
N2 - Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.
AB - Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.
KW - Breast cancer
KW - Immune cells
KW - Immunohistochemistry
KW - Multiplex
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U2 - 10.1007/s10911-021-09479-2
DO - 10.1007/s10911-021-09479-2
M3 - Article
C2 - 33590360
AN - SCOPUS:85099798580
VL - 25
SP - 417
EP - 432
JO - Journal of Mammary Gland Biology and Neoplasia
JF - Journal of Mammary Gland Biology and Neoplasia
SN - 1083-3021
IS - 4
ER -