TY - JOUR
T1 - Automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens
AU - Surace, Michael
AU - DaCosta, Karma
AU - Huntley, Anna
AU - Zhao, Weiguang
AU - Bagnall, Christopher
AU - Brown, Charles
AU - Wang, Chichung
AU - Roman, Kristin
AU - Cann, Jennifer
AU - Lewis, Arthur
AU - Steele, Keith
AU - Rebelatto, Marlon
AU - Parra, Edwin R.
AU - Hoyt, Clifford C.
AU - Rodriguez Canales, Jaime
N1 - Funding Information:
This work was supported by MedImmune, the global biologics R&D arm of AstraZeneca. C.W., K.R., and C.C.H. are employees of Perkin Elmer, which produces the reagents (TSA kit) and multispectral scanners that were used in this work. M.S., K.D., A.H., W.Z., C. Bagnall, C. Brown, J.C., A.L., K.S., M.R., and J.R.C. are employees of MedImmune with stock ownership and/or stock interests or options in AstraZeneca.
Publisher Copyright:
© 2019 Journal of Visualized Experiments.
PY - 2019/1
Y1 - 2019/1
N2 - Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2- phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.
AB - Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2- phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.
KW - Cancer research
KW - Immuno-oncology
KW - Immunohistochemistry
KW - Immunoprofiling
KW - Issue 143
KW - Lung cancer
KW - Multiplex immunofluorescence
KW - Multispectral imaging
UR - http://www.scopus.com/inward/record.url?scp=85061278738&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85061278738&partnerID=8YFLogxK
U2 - 10.3791/58390
DO - 10.3791/58390
M3 - Article
C2 - 30735177
AN - SCOPUS:85061278738
SN - 1940-087X
VL - 2019
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 143
M1 - e58390
ER -