Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

Improved methods are needed to gain insights in how proteins exert their myriad roles in cells and organs. Multiplex in situ proximity ligation assay (misPLA), described herein, can provide a window into the functional states of proteins in cells and tissues by applying pairs of antibody oligonucleotide conjugates to generate amplifiable DNA circles upon proximal binding. The analysis reveals interactions and modifications among sets of proteins, read out by recording the identity and location of the resulting localized DNA amplification products. We applied misPLA to both primary and cultured cells and to formalin-fixated paraffin-embedded (FFPE) tissues, to map dynamic changes in protein localizations, phosphorylations and interactions across surface markers, MAPK, immune-checkpoints, T- and B-cell receptors, and adhesion panels. Comparisons of single-plex versus nineplex assays confirmed that misPLA maintains sensitivity and specificity while increasing throughput and spatial context. Across breast cancer, lymphomas and chronic myeloid leukemia (CML) misPLA uncovered shared and disease-specific signaling patterns, underscoring convergence of oncogenic networks. By preserving tissue architecture and enabling high-content functional spatial proteomics at single-cell resolution, misPLA offers a versatile platform for dissecting signaling heterogeneity, pathway crosstalk, and therapeutic responses, with broad applications in cell biology, biomarker discovery and in precision oncology.