Oncogenic drivers play central roles in tumorigenesis as well as in tumor cell survival and proliferation. Mutations of the epidermal growth factor receptor gene (EGFR) that result in constitutive activation of the receptor tyrosine kinase have been identified as oncogenic drivers in a subset of non-small cell lung cancer (NSCLC). PCR-based assays are usually adopted for the detection of EGFR mutations, but no methods to detect EGFR activation that are not based on mutation identification have been established in the clinical setting. We describe a proximity ligation assay (PLA) used to visualize and quantitate EGFR homodimerization in NSCLC cell lines and tissue specimens. Rabbit monoclonal antibodies against EGFR were conjugated to PLUS or MINUS PLA oligonucleotide arms using Probemaker. Annealing of the PLUS and MINUS PLA probes occurred when two EGFR monomers were in close proximity, and repeat sequences in the annealed oligonucleotide complexes were amplified then recognized by a fluorescently-labeled oligonucleotide probe. PLA signals were detected and counted with a fluorescence microscope. We demonstrate the detection of EGFR homodimers by PLA analysis in a quantitative manner in both NSCLC cell lines and tissue samples obtained by transbronchial lung biopsy. PLA methods are a new tool for the detection and quantitation of protein-protein interactions such as homodimers, heterodimers, and fusion proteins.
Keywords: Oncogenic driver, Epidermal growth factor receptor (EGFR), Anaplastic lymphoma kinase (ALK), Non-small cell lung cancer (NSCLC), Proximity ligation assay (PLA), Dimerization
Activating mutations of the epidermal growth factor receptor gene (EGFR) are currently detected by PCR-based assays ( Nagai et al., 2005 ). However, the visual detection and quantitation of activated EGFR in the clinical setting has not been established. In situ proximity ligation assay (PLA) is a technology that uses Duolink ® In situ reagents (see References 1 and 2) to create probes by conjugating oligonucleotides to antibodies. When two different types of PLA probes (PLUS and MINUS) are in close proximity (40 nm), annealing occurs which generates an amplified circular DNA. The signal from each detected pair of PLA probes is visualized as an individual spot, and the number of PLA signals per cell can be counted with a fluorescence microscope ( Figure 1 ).
A. PLA probes, created by conjugating PLA oligonucleotides and monoclonal antibodies for EGFR, bind to EGFR. B. Connected oligonucleotides hybridize and create multiple circular DNA molecules. C. Fluorescently-labeled detection of oligonucleotides hybridizing to the DNA circle. (Pictures were taken from the Duolink ® In situ User Guide [References 1 and 2]).
The PLA method can be used to detect any protein-protein interactions in close proximity. We have now applied the PLA using primary antibodies derived from the same species to visualize and quantitate EGFR homodimerization. Furthermore, we detected the formation of EGFR-human epidermal growth factor receptor2 heterodimers and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein in NSCLC cell lines ( Ota et al., 2017 ). This method will be applicable to the detection of other dimerizations and fusions.
Standard pipette tips with a volume capacity of 10 μl, 20 μl, 100 μl, 200 μl, and 1,000 μl (Thermo Fisher Scientific, catalog numbers: 2140, 2149P, ART 10REACH, ART 20P)
