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Abstract
Background Formation of tumor immune microenviroment (TIME) and interaction with tumor cells impacts response to immunotherapy. Tyramide-based multiplex immunofluorescence (mIF) on FFPE tumor samples is a powerful technology to study the concomitant expression and localization of immune biomarkers while preserving tissue architecture and spatial context in TIME. 4–1BB, an activation-induced costimulatory molecule for immune cells, is an important regulator response and being actively developed in clinical trials. The current 7 plex opal mIF study targeting 4–1BB aims to characterize the 4–1BB positive TIME, particularly 4–1BB positive B lymphocytes and their spatial relationship with myeloid and tumor cells.
Methods The automated 7-color mIF assay (figure 1) was designed to study expression of 6 biomarkers targeting tumor or immune cells, PanCytokeratin (PanCK), 4–1BB, CD11c, CD68, CD163, CD20 and nuclear counterstain DAPI simultaneously on FFPE tissue sections of head and neck squamous cell carcinoma and non-small cell lung carcinoma. Tonsil FFPE tissue were included for sample and staining controls (figure 2).
PanCK, 4–1BB, CD11c, CD68, CD163 and CD20 primary antibodies were first validated by IHC in tonsil positive control tissue (figures 3 and 4). The staining condition of the 7-color complete TSA amplification Opal mIF assay was then optimized and developed on Leica Bond auto stainer. Assay variation in HNSCC and NSCLC was determined in precision experiment (figures 5 and 6). In brief, 3 individual samples from each disease indication and 9 sequential sections from each sample were used to calculate the cell density for each biomarker and the coefficient of variations (CVs) of cell density were determined among intra/inter experimental runs.
Results The specificity and accuracy of staining patterns of the listed biomarkers were benchmarked in control tonsil and tumor tissues by comparing the positivity and staining patterns between mIF and correspondent singleplex IHC images. Precision assessment was performed to demonstrate reproducibility of the assay. For high prevalence biomarkers, PanCK and CD20, CVs range less than 5–20% were observed in tonsil samples. CVs ranges higher than 5–20% were observed in tumor samples (figures 7–9). For 4–1BB as a less prevalent biomarker, higher CV ranges were observed in both tonsil and tumor samples (figure 9). Interestingly, 4–1BB positive B cells were found to be enriched in the germinal center (GC) of secondary lymphoid follicles in tonsil tissues, and in tertiary lymphoid structures (TLS) in tumor samples.
Conclusions These results indicate that the Opal mIF panel is sufficient to study 4–1BB positive immune cells and their spatial relationship with myeloid and tumor cells in TIMEs.
Schematic figure of 7 plex mlF Opal staining work flow.
Antibody IHC validation on Tonsil.
Optimized Opal and antibody conditions of 7 Plex Opal Panel.
Result 3A. & Plex opal panel on Tonsil. Representative image of germinal center of Tonsil tissue with sinplex biomarker --Pan CK, 41BB, CD20, CD11c, CD68 and CD163.
Result 3B. 7 Plex opal panel on HNSCC. Representative images of biomarker: complete panel --Pan CK, 41BB, CD20, CD11c, CD68 and CD163 in tertiary lymphoid structure (TLS) on head and neck squamous cell carcinoma tissue.
Result 3C. 7 Plex opal panel on Tonsil, HNSCC and NSCLC ADC. Representative images of single biomarker --Pan CK, 41BB, CD20, CD11c, CD68 and CD163 on head and neck squamous cell carcinoma tissue.
Coefficient variant (CV) of CD20 (Opal 620) in Tonsil 1573A and Tonsil 2005A tissues, inter CV9.7%-11.2% and intra CV1.0%-4.3%.
Coefficient variant (CV) of CD20 (Opal 620) in Tonsil 1573A and Tonsil 2005A tissues, inter CV9.7%-11.2% and intra CV1.0%-4.3%.
Overall CVs of all biomarkers in 3 different tumor samples.
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