Developing an enhanced 7-color multiplex IHC protocol to dissect immune infiltration in human cancers

Abstract

The TSA Opal multiplex immunohistochemistry (mIHC) protocol (PerkinElmer) has been used to characterize immune infiltration in human cancers. This technique allows multiple biomarkers to be simultaneously stained in a single tissue section, which helps to elucidate the spatial relationship among individual cell types. We developed and optimized two improved mIHC protocols for a 7-color panel containing 6 biomarkers (CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK)) and DAPI. The only difference between these two protocols was the staining sequence of those 6 biomarkers as the first sequence is PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and the second sequence is FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI. By comparing PD-L1/FoxP3 staining in mIHC and singleplex PD-L1/FoxP3 staining on the adjacent slide, we demonstrated that the staining sequence does not affect the staining intensity of individual biomarkers as long as a proper antigen retrieval method was used. Our study suggests that use of an antigen retrieval buffer with higher pH value (such as Tris-EDTA pH9.0) than that of the stripping buffers (such as citrate buffer pH6.0) is helpful when using this advanced mIHC method to develop panels with multiple biomarkers. Otherwise, individual biomarkers may exhibit different intensities when the staining sequence is changed. By using this protocol, we characterized immune infiltration and PD-L1 expression in head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens. We observed a statistically significant increase in CD3+ cell populations within the stroma of NSCLC as compared to BCa and increased PD-L1+ tumor cells in HNSCC as opposed to BCa.

Spatial Profiles of Intratumoral PD-1+ Helper T Cells Predict Prognosis in Head and Neck Squamous Cell Carcinoma

“…Since immune cells require multiple markers for lineage and phenotype identification, quantitative evaluation of immune cell lineages with spatial information has been technically challenging due to the limited number of analyzable markers in conventional IHC and immunofluorescence methodologies. To tackle those grand challenges, a variety of new approaches have been actively developed in the field of head and neck cancer as well as various malignancies via seminal technologies such as NanoString GeoMx digital spatial profiling (24), Vectra Polaris ( 16), co-detection by indexing (25), Visium spatial gene expression analysis (26) and other mIHC technologies (12,27,28). Owing to recent advances in multiplex IHC and immunofluorescence technologies as well as spatial genomics and molecular imaging technologies, we adopted a 12-marker chromogenic multiplex IHC platform with tissue segmentation algorithms, enabling identification of cellular components with preserved tissue architecture (Figures 1B-D).…”

Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer

“…mIHC takes advantage of multiple cycles of staining and antibody stripping to allow detection of multiple markers on a single tissue section, thereby overcoming the limitation of standard chromogenic IHC. Despite the effectiveness and reliability of mIHC in simultaneously detecting several markers, most of the published mIHC protocols have been optimised for human FFPE specimens [5,31,32]. Since preclinical mouse models are indispensable in translational research, we sought to optimise a protocol and mIHC workflow for mouse FFPE tissue to characterise the immune microenvironment in intestinal disease and cancer.…”

Pan-cancer Analysis Reveals the Small Nuclear Ribonucleoprotein Polypeptide C (SNRPC) Its Significance in Human Cancers

“…Immersion of tumor immune cells as an important part of the tumor microenvironment, and cancer closely related to the occurrence and development of or transfer [20][21][22]. We found that using the TIDE algorithm in the database, SNRPC was found to be related to the inhibition of immune cells from myelin sources of many tumors.…”