Parallel Visualization of Multiple Protein Complexes in Individual Cells in Tumor Tissue

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.A far greater understanding of proteins interacting in complexes in cells and tissues is needed to explain the functional states of cells. Accordingly, there is a pressing need for improved methods to study protein interaction complexes to explain disease mechanisms; however, suitable methods have been lacking, particularly for clinical material. As an example, proteins in the epidermal growth factor receptor (EGFR)¹ family have traditionally been used as clinical markers. However, in many cases this has proven of limited prognostic value, and activity markers such as receptor interactions are attracting increasing interest (1, 2). Methods such as FRET-based detection (3) or the VeraTag assay (4) can be used to investigate protein complex formations in patient tissues. However, such techniques are not suitable to measure several concurrent protein complexes as required to characterize the balance between different segments of a signaling pathway or between different pathways. FRET-based methods are difficult to use with clinical material and have very limited multiplexing capabilities. The VeraTag assay can be multiplexed, but it fails to provide spatial information of the complexes and thus cannot distinguish between cancer cells and surrounding stroma. Similarly, bulk measures of protein complexes via e.g. co-immunoprecipitation and mass spectrometry (5) disregard cell-to-cell variations and the subcellular distributions of protein complexes. Moreover, such methods are poorly suited for analyzing precious clinical material as too much sample material is needed for the analysis.To enable parallel analyses directly in tumor tissue of multiple protein complexes involved in signaling pathways, we have developed a multiplex version of the in situ proximity ligation assay (PLA)¹ (6). In situ PLA has previously been used for localized detection of proteins, protein complexes, and post-translational modifications in cells and tissues (6). Because of its intrinsic requirement for dual target recognition by pairs of antibodies and the use of rolling circle amplification (RCA) to substantially amplify signals, the assay allows detection of endogenous protein complexes or post-translational modifications in fixed cells and tissue sections (7, 8) or Western blot membranes (9). The basis of in situ PLA is the detection of a target molecule through the use of a pair of PLA probes, i.e. target-specific affinity reagents such as antibodies to which DNA oligonucleotides have been attached (Fig. 1). We describe herein how tag sequences in the oligonucleotides of each PLA probe, uniquely identifying these probes, can be propagated into the single-stranded RCA products that result when two PLA probes have bound complex-forming proteins. The amplified tags in the RCA products can then 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.Open in a separate windowFig. 1.Parallel detection of protein complexes using multiplex in situ PLA. Groups of PLA probes are used to detect all binary complexes between a protein X and any of the proteins A–C. Using oligonucleotides attached to specific antibodies as templates, two linear connector oligonucleotides and one probe-specific tag oligonucleotide are enzymatically joined into a DNA circle that subsequently templates RCA. The RCA products, whose repeated sequences identify the protein in complex with protein X, can be visualized by hybridization of three tag-specific detection oligonucleotides labeled with distinct fluorophores.To test our probe design, we targeted the well characterized EGFR family. This family consists of four transmembrane tyrosine kinase receptors (EGFR, HER2, HER3, and HER4), involved in the regulation of fundamental cellular functions such as cell growth, survival, death, differentiation, and proliferation (10). Increased expression, or aberrant regulation, of the receptors has been implicated in a broad range of human malignancies, including breast cancer, where overexpression of HER2 is associated with a poor prognosis (11). Members of the EGFR family can interact in different constellations, with 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 (2–4, 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.