In Situ detection of protein-phosphoinositide signalosome in tissues by immunohistochemistry-coupled proximity ligation assay

Abstract

Abstract Phosphoinositides (PIPs) are central lipid signaling molecules, with PtdIns(3,4,5)P 3 acting as a key regulator of the PI3K-AKT pathway and a major driver of tumorigenesis. Together with p53 mutations or loss, this pathway represents one of the most frequently dysregulated axes across human cancers. We recently identified a nuclear p53-PtdIns(3,4,5)P 3 signalosome that enables de novo AKT activation, promoting cancer cell survival, invasion, and chemoresistance, with potential biological and clinical relevance. However, in situ detection of such protein-lipid signalosome in tissues has remained technically challenging. Here, we present an immunohistochemistry-coupled proximity ligation assay (IHC-PLA) that integrates PIP-specific antibodies with proximity ligation assay (PLA) and immunohistochemistry to visualize endogenous PtdIns(3,4,5)P 3 -mutant p53 interaction in breast cancer tissues. This approach enables semi-quantitative, high-resolution detection of nuclear protein-PIP signalosome in situ , providing a versatile tool for interrogating PIP signaling in cancer pathology.