Abstract
Cellular functions are regulated and executed by complex protein interaction networks. Accordingly, it is essential to understand the interplay between proteins in determining the activity status of signaling cascades. New methods are therefore required to provide information on different protein interaction events at the single cell level in heterogeneous cell populations such as in tissue sections. Here, we describe a multiplex proximity ligation assay for simultaneous visualization of multiple protein complexes in situ. The assay is an enhancement of the original proximity ligation assay, and it is based on using proximity probes labeled with unique tag sequences that can be used to read out which probes, from a pool of probes, have bound a certain protein complex. Using this approach, it is possible to gain information on the constituents of different protein complexes, the subcellular location of the complexes, and how the balance between different complex constituents can change between normal and malignant cells, for example. As a proof of concept, we used the assay to simultaneously visualize multiple protein complexes involving EGFR, HER2, and HER3 homo- and heterodimers on a single-cell level in breast cancer tissue sections. The ability to study several protein complex formations concurrently at single cell resolution could be of great potential for a systems understanding, paving the way for improved disease diagnostics and possibilities for drug development.
Highlights
A far greater understanding of proteins interacting in complexes in cells and tissues is needed to explain the functional states of cells
We describe how tag sequences in the oligonucleotides of each PLA probe, uniquely identifying these probes, 1 The abbreviations used are: epidermal growth factor receptor (EGFR), EGF receptor; PLA, proximity ligation assay; RCA, rolling circle amplification
To enable multiplex combinatorial in situ PLA, we included a tag-specific sequence within the PLA probe
Summary
Visualization of Multiple Protein Complexes in Tumor Tissue connector oligonucleotides “A” tag protein X protein A “B” tag protein X protein B “C” tag protein X protein C. The amplified tags in the RCA products can be visualized using detection oligonucleotides, labeled with different fluorophores, to uniquely recognize the tag sequences. This multiplex readout makes it possible to compare levels of protein complexes between individual cells by identifying the PLA probes that gave rise to the signals. HER2 as the preferred interaction partner [12], activating several signaling pathways These interactions between different members of the EGFR family and with associated proteins have been studied extensively in many different types of cells and tissues with a range of methods [2,3,4, 13], including in situ PLA (14 –17). Using multiplex in situ PLA, we successfully visualized multiple protein complexes in cultured cells and in fresh frozen tissue sections, illustrating the potential to study the balance between alternative protein complexes in clinical specimens to identify cellular phenotypes
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