Table 1.
Antibodies tested for developing the mIHC protocols.
Fig 1.
Anti-CD163, CK, and PD-L1 were validated using the vendor’s provided antigen retrieval method Tis-EDTA (pH 9.0) as an antigen retrieval buffer (A, B, C). Anti-CD8, CD3, and FoxP3 were validated using the vendor’s suggested citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) in adjacent tissue sections (D-I). The frequency (mean+/-SD) of each biomarker was determined (D-I).
Fig 2.
Chromogenic vs. Opal: Validation of singleplex Opal fluorescent stains in human FFPE tonsil tissue specimens.
The optimized titrations of primary antibodies (CD8, CD163, CD3, FoxP3, PD-L1, and CK) which used Tris-EDTA pH9.0 to retrieve antigens in singlepex chromogenic stain were transferred to Opal fluorescent stain on the adjacent slides. The frequency (mean+/-SD) of each biomarker was determined.
Fig 3.
Comparison of staining sequences for mIHC.
Schematic diagrams show two protocols with different staining sequences (A). Multiplex IHC and single color images were obtained using these two protocols from human tonsil (FFPE) tissue serial sections. H&E images show the morphology (B, C). Singleplex IHC in adjacent slides verified individual biomarker stains in mIHC (B, C). The mIHC stain followed Protocol 1 in (B) and Protocol 2 in (C).
Fig 4.
Quantification analysis on PD-L1+, FoxP3 +, CD3+CD8+, and CD3+FoxP3+ cell populations.
Quantification analysis confirmed the percent PD-L1+ cell population determined by mIHC is comparable with singleplex IHC in the adjacent sections (A-D). The threshold of PD-L1+ cell populations was determined by a Board-certificated Pathologist. The staining intensity of PD-L1 cells above the threshold were recognized as positive cells. The mIHC stain followed Protocol 1 in (A) and Protocol 2 in (C). Similar analysis was conducted with respect to FoxP3 expression (E, F). CD3+CD8+ and CD3+FoxP3+ cell populations stained by Protocol 1 and Protocol 2 were analyzed (G, H).
Fig 5.
mIHC staining and analysis of immune infiltration of HNSCC, NSCLC, and BCa specimens.
The protocol was validated in head and neck cancer (HNSCC), non-small cell lung cancer (NSCLC), and breast cancer (BCa) patient samples. H&E images show the morphology (A). Different cell populations were compared among different cancer types including T cells (CD3+ or CD8+), Treg (FoxP3+), and PD-L1+ tumor cells. The median of each patient from all available ROI was used for analysis (B, C). Graphs depict the mean+/-range from all patients within each cohort. **P<0.01 (1-way ANOVA).
Table 2.
Patient demographics.