Abstract 43P
Background
The study of tumor microenvironment (TME) has become more and more important over the past few decades as it plays a critical role in cancer development. Studies have been focusing on investigating the immune components within TME as prediction biomarkers to increase immunotherapy efficacy and to assist patient stratification for precision immune therapy due to single biomarkers’ inability to stratify the different therapy strategies, in this study we employed the multiplex immunohistochemistry (multiplex-IHC) to illustrate multiple biomarkers within TME in single tissue section and explore the immune contexture in non-small cell lung carcinoma (NSCLC). Due to antigen properties, the different antigen retrieval solutions, the concentration of detecting antibodies, and staining sequential on each staining step in multiplex IHC will affect the efficacy of staining outcome, therefore, optimization of those staining conditions is necessary for generating accurate results.
Methods
CD3, PD-1 and PD-L1 were selected as biomarkers to verify the immune components within TME by multiplex-IHC on single tissue section. Antigen retrieval solutions, Citrate pH 6.1 (DAKO®) and EDTA buffer (Thermo®), were tested for each staining.
Results
After applying different antigen retrieval solutions of IHC staining, we found out that the optimized antigen retrieval solution for PD-1 and PD-L1 was EDTA buffer (Thermo®). For dilution of detecting antibodies, we concluded that 1:1000 CD3 (DAKO), 1:200 PD-1 (CST) and 1:500 PD-L1 (CST) as the best conditions for multiplex-IHC. In addition, the optimized staining order could be PD-L1 (cycle 1), CD3 (cycle 2) and PD-1 (cycle 3). The next step would be to apply suitable myeloid and lymphoid markers on adjacent slides with Opal™ 7 color kits to discover the immune microenvironment of NSCLC.
Conclusions
Our experimental results show the ability of multiplex-IHC to verify the immune contexture in NSCLC. This work can not only investigate the immune contexture in TME for pre-clinical immuno-oncology study, but also can apply to personalized medicine and clinical cancer treatment.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Ministry of Science and Technology, Taiwan, R.O.C.
Disclosure
All authors have declared no conflicts of interest.