Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease.This research was funded by the Prostate Cancer Research Centre charity (registered UK charity no. 1156027), Grant Number AA1. A small financial contribution was also made through intra-mural funds from the Royal Veterinary College.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.016110

Multi-labeled immunofluorescence for CD1, c-Myc and FRA1.

<p>Co-expression of three proteins CD1 (FITC-green), c-Myc (Cy3-red) and FRA1 (Cy5-cyan) in normal (A, B and C) and malignant feline oral tissue cores (D, E and F) and DAPI, counterstain is blue; images are representative tissue cores from the tissue array with over 200 samples. Whole tissue cores (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#pone.0161103.s004" target="_blank">S4 Fig</a>) were imaged using a Zeiss Axioscan Z.1 slide scanner (Carl Zeiss) at 20x magnification with a Calibri.2 LED lights and integration times of the Hamamatsu ORCA Flash4 camera (Hamamatsu Photonics) and fluorescent signals were optimized at the start of study so as to not oversaturate the signal for each antibody. For quantitative co-localization a randomly selected area was imaged using an Olympus IX81 confocal system and a dry 40x objective (A, normal and D, malignant); these areas were further magnified (6x digital zoom) and re-imaged (B, normal and E, malignant). The resulting images were deconvolved using Huygens Software (C, normal and F, malignant) for the calculation of GIC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#sec002" target="_blank">Methods</a>). Scale bar = 10μm.</p

Quantitation of DAB signal in feline tissue array.

<p>Total Area (TA) stained (in pixel) with each antibody was quantified using ImageJ software using an unbiased, automated protocol (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#sec002" target="_blank">Methods</a>); TA is converted to Area Fraction (AF, total area divided by the total pixels in the image). Each bin is data for an individual, malignant (red) or normal tissue (green) core. There is a significant increase in the AUC of protein expression in malignant vs normal feline tissue (p<0.0001).</p

ROC of putative Wnt related proteins in FOSCC.

<p>ROC curve demonstrates the discriminating performance of the protein expression in the differentiation between malignant and normal tissue cores using the area fraction (probit) data for (A) CD1, (B) c-Myc and (C) FRA1. The operating characteristic values are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#pone.0161103.s007" target="_blank">S1 Table</a>. The dotted line represents the ROC area of 0.5.</p

Box plots for differential co-localization of CD1, c-MYC and FRA1 in feline tissue samples.

<p>High magnification fluorescent images (e.g. as represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#pone.0161103.g004" target="_blank">Fig 4</a>) of feline tissue cores (normal, gray outline; squamous cell carcinoma, brown outlined boxes) were deconvolved and used for the calculation of Global Intersection Coefficient (GIC) using Huygens software. 19 individual tissue samples were used for the three proteins and nuclear stain DAPI (DP) to measure the co-localization of DP/CD1 (blue/green bars), DP/c-MYC (blue/red), DP/FRA1 (blue/cyan), CD1/c-MYC (green/red), CD1/FRA1 (green/cyan) and c-MYC/FRA1 (red/cyan) using GIC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161103#sec002" target="_blank">Materials and Methods</a>). Significance of difference in the GIC for co-localization between normal and FOSCC was calculated using the Mann Whitney U test (* = <i>p</i><0.01, ns = not significant).</p

Quantitative analysis of protein expression in FOSCC.

<p>Quantitative analysis of protein expression in FOSCC.</p