TY - JOUR
T1 - Immuno-profiling and cellular spatial analysis using five immune oncology multiplex immunofluorescence panels for paraffin tumor tissue
AU - Parra, Edwin Roger
AU - Ferrufino-Schmidt, Maria C.
AU - Tamegnon, Auriole
AU - Zhang, Jiexin
AU - Solis, Luisa
AU - Jiang, Mei
AU - Ibarguen, Heladio
AU - Haymaker, Cara
AU - Lee, J. Jack
AU - Bernatchez, Chantale
AU - Wistuba, Ignacio Ivan
N1 - Funding Information:
This study was supported in part by the scientific and financial support for the CIMAC-CIDC Network provided through the National Cancer Institute (NCI) Cooperative Agreement U24CA224285 of the MD Anderson Cancer Center CIMAC and for the Translational Molecular Pathology Immunoprofiling Laboratory, National Institutes of Health/NCI through Cancer Center Support Grant P30CA016672 (used the Institutional Tissue Bank), and SPORE grant 5P50CA070907-18, by the Cancer Prevention and Research Institute of Texas through MIRA RP160688.
Funding Information:
The Translational Molecular Pathology Immunoprofiling Laboratory thanks their members, who contribute daily to quality mIF and IHC staining. To our data analysts, Renganayaki Krishna Pandurengan and Shanyu Zhang. To Jose de Jesus Martins, PhD, mathematician, for teaching me the different R-studio tools for performance the spatial analysis in this project. Editorial support was provided by Sarah Bronson and Bryan Tutt of Editing Services, Research Medical Library, The University of Texas MD Anderson Cancer Center.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-021-88156-0
DO - 10.1038/s41598-021-88156-0
M3 - Article
C2 - 33875760
AN - SCOPUS:85104505733
SN - 2045-2322
VL - 11
JO - Scientific reports
JF - Scientific reports
IS - 1
M1 - 8511
ER -