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PLoS One. 2016 Aug 25;11(8):e0161103. doi: 10.1371/journal.pone.0161103. eCollection 2016.

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

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Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom.
Department of Clinical Sciences and Services, The Royal Veterinary College, London, United Kingdom.
Division of Surgery, University College London, London, United Kingdom.
Prostate Cancer Research Center at the Centre for Stem Cells and Regenerative Medicine, King's College London, London, United Kingdom.
Department of Veterinary Sciences, University of Pisa, Pisa, Italy.
IDEXX Laboratories Ltd., Grange House, West Yorkshire, United Kingdom.
Dundee Imaging Facility, College of Life Sciences, University of Dundee, Dundee, United Kingdom.
Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.
Department of Pathology and Pathogen Biology, The Royal Veterinary College, London, United Kingdom.
Department of Comparative Biomedical Sciences, The Royal Veterinary College, London, United Kingdom.
University of London, London, United Kingdom.
Independent Vetcare, Bath, United Kingdom.
Research Department of Cell and Developmental Biology, The Centre for Cell and Molecular Dynamics, Rockefeller Building, University College London, London, United Kingdom.


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.

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Conflict of interest statement

Competing Interests: The authors of this manuscript have the following competing interests: AA (principal inventor) and CT and JRM (co-inventors) have filed for a patent through UCL Business for targeting Wnt signaling pathway for ß-catenin related disorders (New British Patent Application no. 1318659.8). AA is an associate editor of PLOS ONE. This do not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. One co-author (Jenny McKay) works for a commercial organization called IDEXX; another co-author (Malcolm Weetman) works for a commercial organization Independent Vetcare. The corresponding author declares, categorically, that none of the issues stated in these statements alter the authors' adherence to all policies of PLOS ONE.

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