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

Area of Science:

• Oncology
• Immunology
• Biotechnology

Background:

• Multiplex immunohistochemistry (mIHC) characterizes immune infiltration in human cancers by simultaneously staining multiple biomarkers.
• Optimizing mIHC protocols is crucial for accurately elucidating spatial relationships among immune cell types within the tumor microenvironment.

Purpose of the Study:

• To develop and optimize two improved mIHC protocols for a 7-color panel including CD3, CD8, CD163, PD-L1, FoxP3, and cytokeratin (CK).
• To assess the impact of biomarker staining sequence on staining intensity and validate the protocol for cancer immune profiling.

Main Methods:

• Developed two mIHC protocols with different biomarker staining sequences (PD-L1/CD163/CD8/CK/CD3/FoxP3/DAPI and FoxP3/CD163/CD8/CK/CD3/PD-L1/DAPI).
• Utilized antigen retrieval buffers with higher pH (Tris-EDTA pH9.0) compared to stripping buffers (citrate buffer pH6.0).
• Validated staining consistency by comparing mIHC with singleplex staining and applied the protocol to head and neck squamous cell carcinoma (HNSCC), breast cancer (BCa), and non-small cell lung cancer (NSCLC) specimens.

Main Results:

• Demonstrated that staining sequence does not affect biomarker intensity with proper antigen retrieval.
• Identified statistically significant increases in CD3+ cells in NSCLC stroma compared to BCa.
• Observed increased PD-L1+ tumor cells in HNSCC compared to BCa.

Conclusions:

• Optimized mIHC protocols, particularly with appropriate antigen retrieval (higher pH buffers), ensure reliable biomarker detection.
• The developed protocol effectively characterizes immune infiltration and PD-L1 expression across different cancer types (HNSCC, BCa, NSCLC).
• Findings highlight distinct immune profiles in NSCLC and HNSCC, providing insights for targeted therapies.