High-throughput optical proteomics and breast cancer patient profiling: novel applications to individualise prognosis and treatment

Background

Breast cancers that appear similar by stage and grade are not identical in terms of outcome for each patient affected. Heterogeneity would be better understood using genomic/proteomic profiles to predict for relapse. Risk estimation could be truly individualised and treatment personalised. Proteomics goes beyond possession of an abberrant gene by assessing post-translational modifications such as phosphorylation and measuring protein–protein interactions. Optical proteomics uses fluorescence lifetime imaging microscopy (FLIM) to quantify associations between signalling proteins in tissues beyond the spatial resolution of light microscopy by measuring Förster resonance energy transfer (FRET). These technologies improve understanding of how extracellular signals are sensed by cancer cells and transduced to trigger invasion. Protein kinase C alpha (PKCα) is a signalling protein that can oppose apoptosis. The actin-binding protein ezrin provides a direct link between the cytoskeleton and plasma membrane, necessary for cell migration and metastasis. Ezrin–PKCα interaction has been demonstrated in breast cancer cell line experiments [1].

Methods

Fluorophore-conjugated antibodies to PKCα and ezrin were applied to breast cancer tissue microarrays (TMA), obtained from a well annotated tissue bank with a rich complement of clinical data. Each TMA was created from 84 invasive breast carcinoma samples, formalin fixed and paraffin embedded. Immunofluorescence enables imaging of both proteins simultaneously at two different wavelengths from the same section of tissue. As intensity is proportional to concentration, proteins can be accurately quantified. FLIM analysis was performed. Where anti-ezrin-Cy2 and anti-PKCα-Cy3 are located within nanometre proximity intracellularly, measurable energy transfer occurs (FRET). Controls were matched tumour areas of noninteracting proteins.

Results

We imaged six TMAs (histopathological grades I to III) in triplicate to generate epifluorescence images and FRET/FLIM data. We have demonstrated a wide spectrum of distribution for both ezrin and PKCα in human breast cancer tissue. We have reported on the activation state of ezrin and the colocalisation of both proteins (Figure 1). We have measured FRET between anti-ezrin-Cy2 and anti-PKCα-Cy3. The present study is the first to demonstrate ezrin-PKCα interaction in human breast tissue (Figure 2). In a subset, the FLIM assay was complemented by an independent intensity method. Tissue data are further corroborated by parallel assays performed on cultured cancer cells. All parameters are undergoing multivariate analysis and further statistical comparison with respect to time to relapse of disease.

Figure 1

Ezrin and PKCα colocalised in normal breast ducts. Ezrin–PKCα colocalisation is demonstrated but with a long fluorescent lifetime and a blue pixel-by-pixel fluorescence lifetime (τ) map. The fluorescent lifetime of Cy2 does not reduce. There is no interaction between the proteins. Controls were matched tumour areas in the same tissue core (3 to 5 μm deeper in the TMA block) stained for noninteracting proteins.

Figure 2

Ezrin and PKCα colocalised in grade II invasive breast cancer. The fluorescent lifetime has shortened and a the pixel-by-pixel fluorescence lifetime (τ) map shows areas of red. There is interaction between the proteins in these areas when compared with the control. In this section of invasive ductal breast cancer, ezrin–PKCα interaction is demonstrated as FRET has occurred.

Conclusion

The present study has established several optics-based parameters to be used in a multivariate correlation with breast cancer patient outcome. The goal is to derive multiple high-throughput optical proteomic markers that could be applied to tumour tissue at first diagnosis to better predict risk for individual patients. This project aims to translate advanced optical proteomic science into real-life benefit, assisting patients and physicians in the difficult decisions regarding treatment.