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

Abstract

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.

Telomerase Reverse Transcriptase (TERT) Expression, Telomerase Activity, and Expression of Matrix Metalloproteinases (MMP)-1/-2/-9 in Feline Oral Squamous Cell Carcinoma Cell Lines Associated With Felis catus Papillomavirus Type-2 Infection

“…This might be plausible also in FOSCC, since SCCF3 harboring higher cMyc amounts had displayed lower sensitivity to several chemotherapeutics with respect to SCCF2, as described in a recent work (45). A possible role of cMyc in FOSCC development was further suggested in an elder report showing its overexpression in tumor samples, consistently with our results (46).…”

Equine penile squamous cell carcinoma: expression of biomarker proteins and EcPV2

“…A sample size calculation was made incorporating parameters from previously described studies 18,[28][29][30] ; the minimum tumour and control group sizes were 21 and 7 respectively to ensure adequate statistical power to detect differential expression of protein signal. Tissue arrays were constructed using a Beecher Instruments Microarrayer (MTA-1 tissue arrayer, Beechers Instruments, Sun Prairie, WI, USA).…”

“…In analysing cancer related proteins, putatively associated with the Wnt pathway, in EpSCC and their association with EcPV2 infection we may find dual benefit in creating an animal model of human disease as well as improving our understanding of an equine condition that causes significant morbidity. Using a cohort of EpSCC tissue, we conducted medium throughput protein staining and imaging and a quantitative approach previously established in our laboratory 18,[28][29][30][31] to investigate the hypothesis that viral-driven alterations in inflammation may play a role in EpSCC development.…”

Expression of ribosomal proteins in normal and cancerous human prostate tissue

“…Quantification of Ox-DAB staining was achieved by first obtaining high resolution images of each tissue core on a bright field microscope (Nikon) for particle analysis with ImageJ, as described previously [ 20 ]; imaging tiles of the whole tissue array were also obtained using a Nano-zoomer (Hamamatsu) for illustrative purposes. The amount of tissue present in each image was calculated using a sequence of macros [ 7 , 8 ] to: open image, invert image, convert image to 16bit, select a predetermined threshold selected using a random training set, convert image to mask, analyse particles (Size 0.5-Infinity, Circularity 0.00–1.00), save then close the image. A similar macro was then constructed to measure the number of particles expressing Ox-DAB signal in the segmented pixels.…”

“…Gene or protein biomarkers in the biopsy sample could assist in addressing some of these issues. We have previously used largescale tissue arrays to identify proteins that are differentially expressed in non-malignant vs malignant samples using a reproducible and quantitative approach [ 7 , 8 ], rather than utilising a subjective interpretation of immunohistochemical expression. In the current study, we have applied the quantitative approach to a tissue array containing 245 human prostate tissue samples, gathered from 82 patients, stained for ribosomal (RPS) proteins.…”