Visiting Fellowship – Report on ‘Development of the novel in-situ proximity ligation assay (PLA) for use in quantifying c-Met receptor activation’
By Dr. Colan Ho-Yen, MBChB, FRCPath (CR-UK Clinical Research Fellow, Barts Cancer Institute)
PhD supervisors: Prof. Louise Jones and Dr. Stéphanie Kermorgant
Visiting supervisor: Assoc. Prof. Ola Söderberg, Rudbeck laboratory, Uppsala University, Sweden
29th April 2012 – 8th May 2012
Background:
Over-expression of the receptor tyrosine kinase c-Met is implicated in breast cancer progression and there are numerous ongoing clinical trials assessing the efficacy of c-Met inhibition in a variety of cancers1. Quantifying the activity of the receptor may give more clinically relevant information than simple protein expression and the PLA offers the potential to accomplish this in an in-situ setting.
The proximity ligation assay (PLA) is designed to detect proteins, protein-protein interactions and post-translational modifications, utilising two antibodies linked to short DNA strands. When the antibodies bind in close proximity, a circle of DNA is formed, which acts as a template for a rolling circle amplification step (RCA). The amplified DNA can then be visualized under fluorescent microscopy following the addition of fluorescently-labeled oligonucleotides2. The PLA has several advantages over immunohistochemistry: 1) the requirement for two antibodies to bind increases specificity, 2) the presence of an amplification step enhances sensitivity and 3) the nature of the reaction product allows automated quantification3. It is expected that PLA could in the future be used for diagnosis, prognosis or to predict response to therapy.
Aim: The overall aim of my PhD is to characterise the clinical and functional significance of c-Met in aggressive breast cancers in women of different ethnic backgrounds. Specifically, the aim of this visiting fellowship was to gain experience in using the PLA technique to analyse c-Met activity in cells and tissues, in a laboratory with considerable expertise in the development and optimisation of the assay.
Learning outcomes:
1) I gained practical experience in performing the PLA, on a variety of different sample types including cells, cell pellets (see Figure 1) and tissue microarrays generated from formalin-fixed, paraffin embedded sections.
Figure 1: MDA-MB-468 cells (in pellet form) following HGF stimulation (blue - DAPI, green – phalloidin, red – PLA product (anti-c-Met and anti-phosphotyrosine primary antibodies) ; x63 objective under oil immersion).
2) I was taught the fundamentals of image capture and quantitative analysis of the PLA
product, including approaches to optimise automated image analysis.
3) I observed and contributed to the conjugation of oligonucleotides to secondary species-specific antibodies.
4) I gained valuable insight into the various adjustments that can be utilised to optimise the PLA, based on the product characteristics of control material.
In general, I feel the fellowship was an invaluable opportunity to gain a unique understanding of the practical and theoretical aspects of the PLA and it has provided me with a sound platform to build on as I optimise the technique in my native laboratory.
Future work: I plan to use the skills and knowledge acquired during my fellowship to apply the PLA to my clinical samples, allowing me to analyse the relationship between tumour c-Met activity and prognostic factors and ethnicity.
References:
1) Gastaldi S, Comoglio PM, Trusolino L. The Met oncogene and basal-like breast cancer: another culprit to watch out for? Breast Cancer Res. 2010;12(4):208. Epub 2010 Aug 23.
2) Söderberg O, Gullberg M, Jarvius M et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods. 2006 Dec;3(12):995-1000.
3) Blokzijl A, Friedman M, Pontén F, Landegren U. Profiling protein expression and Interactions: proximity ligation as a tool for personalized medicine. J Intern Med. 2010 Sep;268(3):232-4.