Pseudoimmunofluorescent immunohistochemistry image analysis of phosphorylated signaling proteins in human esophageal squamous cell carcinoma tissue

Immunohistochemistry is primarily employed to visualize the localization of specific molecules in tissue samples. However, there is an increasing need for software-assisted quantitative assessment. In the present study, we performed inverted blue channel-based pseudoimmunofluorescence image analysis using original immunohistochemistry images. In human esophageal squamous cell carcinoma tissues, various humoral factors promote the phosphorylation of signaling proteins, including protein kinase B (Akt) and/or extracellular signal-regulated kinase 1/2 (ERK1/2), leading to tumor progression. Our method demonstrated applicability in the analysis of localized signaling proteins in histological sections. Relatively high phosphorylated Akt (p-Akt) intensity was observed in the cancer-stroma adjacent (Adj) and noncancerous regions of the superficial layer (SL). Furthermore, localized phosphorylated ERK1/2 (Thr202/Tyr204) was observed in the Adj of the SL and invasive front, distinct from the pattern of p-Akt (Ser473) and p-Akt (Thr308). In conclusion, pseudoimmunofluorescent immunohistochemistry image analysis is useful for the quantitative assessment and objective interpretation of localized signaling proteins in esophageal squamous cell carcinoma. The method can also be applied to analyze various immunohistochemistry images from diverse tissues.