Feline Squamous Cell Carcinoma Research Articles

Histologic, immunohistochemical, and in situ hybridization study of myxoid stroma in feline oral squamous cell carcinoma.

Oral squamous cell carcinoma (oSCC) is a highly invasive malignant neoplasm in cats. Recently, tumor stroma, known as tumor microenvironments, have been considered to play an essential role in tumor progression. However, their role in feline squamous cell carcinoma (SCC) remains unclear. This study aimed to reveal the cancer microenvironment of feline oSCC and evaluate the pathological mechanisms of progression. We used 19 samples from 17 cats with oSCC, which were examined using light microscopy, immunohistochemistry, and in situ hybridization (RNAscope®). Feline oSCCs had two types of stroma, namely fibrotic and myxoid stromal reaction patterns, which were easily distinguished using hematoxylin-eosin staining. The myxoid stroma was rich in hyaluronic acid, which seems to be produced by neoplastic cells. Furthermore, the presence of myxoid stroma was correlated with histological parameters, including the appearance of cancer-associated fibroblasts and tumor budding. Periostin protein expression was also frequently observed in the stroma of feline oSCC and was significantly more common in the myxoid stromal reaction pattern group than in the fibrotic group. Positive signals for periostin mRNA were detected in stromal cancer-associated fibroblasts. This study indicates that the interaction between neoplastic cells and stromal reaction pattern components, such as hyaluronic acid and periostin, may be involved in tumor malignancy. Therefore, we propose that focus be placed not only on the tumor tissue but also on the characterization of the stroma for analyzing feline oSCC.

Molecular characterization of feline immune checkpoint molecules and establishment of PD-L1 immunohistochemistry for feline tumors.

Spontaneous tumors are a major cause of death in cats. Treatment of human tumors has progressed dramatically in the past decade, partly due to the success of immunotherapies using immune checkpoint inhibitors, such as anti-programmed death 1 (PD-1) and anti-PD-ligand 1 (PD-L1) antibodies. However, little is known about the PD-1 pathway and its association with tumor disease in cats. This study investigated the applicability of anti-PD-1/PD-L1 therapy in feline tumors. We first determined the complete coding sequence of feline PD-L1 and PD-L2, and found that the deduced amino acid sequences of feline PD-L1/PD-L2 share high sequence identities (66-83%) with orthologs in other mammalian species. We prepared recombinant feline PD-1, PD-L1, and PD-L2 proteins and confirmed receptor-ligand binding between PD-1 and PD-L1/PD-L2 using flow cytometry. Next, we established an anti-feline PD-L1 monoclonal antibody (clone CL1Mab-7) to analyze the expression of PD-L1. Flow cytometry using CL1Mab-7 revealed the cell surface expression of PD-L1 in a feline macrophage (Fcwf-4) and five mammary adenocarcinoma cell lines (FKNp, FMCm, FYMp, FONp, and FONm), and showed that PD-L1 expression was upregulated by interferon-γ stimulation. Finally, immunohistochemistry using CL1Mab-7 also showed PD-L1 expression in feline squamous cell carcinoma (5/5, 100%), mammary adenocarcinoma (4/5, 80%), fibrosarcoma (5/5, 100%), and renal cell carcinoma (2/2, 100%) tissues. Our results strongly encourage further investigations of the PD-1/PD-L1 pathway as a potential therapeutic target for feline tumors.

A Retrospective Multicentric Study of Electrochemotherapy in the Treatment of Feline Nasal Planum Squamous Cell Carcinoma.

Feline squamous cell carcinoma (SCC) is currently treated with surgery, radiation therapy and electrochemotherapy (ECT). Both the efficacy and/or safety of ECT were evaluated as a sole therapy with bleomycin to treat feline nasal planum SCC (npSCC). Sixty-one cats were enrolled. Local treatment response was evaluated as complete remission (CR), partial remission (PR) or stable disease (SD). Recurrence rate (RR), disease-free interval (DFI) and progression free survival (PFS) were calculated. A six-point scale was used for ECT toxicity. The median tumor size was 1.5 cm. CR was achieved in 65.6% of cases, PR in 31.1% and SD in 3.3%. The overall response rate was 96.7%, RR was 22.5%, median DFI was 136 days, and median PFS was 65.5 days. ECT toxicity was ≤2 in 51% of cats. Tumor recurrence/progression (p = 0.014) and local treatment response (PR: p < 0.001; SD: p < 0.001) influenced survival time. Cats with toxicity >2 showed a higher probability of tumor recurrence/progression. Tumor-related death was higher in cats with PR (p < 0.001) and recurrence/progression (p = 0.002), in ECT treatment with 1 Hz (p = 0.035) and 1200 V/cm (p = 0.011) or 1300 V/cm (p = 0.016). Tumor size influenced local treatment response (p = 0.008) and toxicity (p < 0.001). ECT is an effective treatment for feline npSCCs and should be considered as the first-line procedure for low-stage tumors.

Pilot study evaluating stereotactic body radiation therapy for feline facial squamous cell carcinomas.

The use of stereotactic body radiation therapy (SBRT) has not been assessed in advanced-stage feline solar-induced facial squamous cell carcinomas (SCCs). The objective of this study was to provide preliminary data on the use and safety profile of coarse fractions administered with an SBRT regime to manage advanced-stage feline solar-induced facial SCCs. This retrospective study assessed five cats diagnosed with advanced-stage solar-induced facial SCCs that received SBRT as their primary treatment or, in one cat, following failed surgical intervention. Tumour sites received three fractions totalling 26.25-27 Gy over a 3-5-day period. All patients developed acute effects following SBRT including alopecia, epilation and erythema. Late effects were mild and included alopecia, variable pigmentation and leukotrichia within radiation fields. All patients were alive at the time of article submission, with overall survival times ranging from 118 to 991 days. The results suggest that coarse fractions administered with an SBRT technique is a safe and effective treatment tool for the management of advanced-stage feline solar-induced facial SCCs. These data provide preliminary evidence to support larger, prospective studies evaluating the management of feline facial SCCs with SBRT.

Detection of Epithelial Cell Adhesion Molecule in Feline Normal and Tumor Cell Lines and Tissues With Selected Commercial Anti-human EpCAM Antibodies.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane protein expressed at intercellular junctions in epithelial cells. As an epithelial biomarker, it used for immunologic-based capture of epithelial-derived circulating tumor cells (CTCs) in human patients with different carcinomas. EpCAM expression has not been described in normal or neoplastic epithelial tissues in cats. Our goal was to find a commercial antibody that recognizes surface EpCAM expression for CTC detection. We tested two anti-human EpCAM antibodies, designated for use with flow cytometry, for detection of surface EpCAM expression on feline cell lines derived from normal mammary and renal epithelia and mammary and oropharyngeal squamous cell carcinomas in cats. Only one of the antibodies, a goat polyclonal antibody, labeled normal and neoplastic feline mammary epithelial cells and oropharyngeal squamous cell carcinoma cells; no labeling was observed for normal feline kidney epithelial cells. At low dilution, this antibody immunohistochemically stained the intercellular junctions of normal pancreatic, intestinal and mammary epithelium, as well as neoplastic mammary epithelium in feline tissues; however, oral mucosa, skin, and an oropharyngeal squamous cell carcinoma showed no positive immunostaining. The antibody only weakly bound feline squamous cell carcinoma cell lines under static adhesion. Our results indicate that EpCAM is expressed in specific epithelia in cats but is variably expressed in feline mammary tumors and oropharyngeal squamous cell carcinoma. A higher avidity cross-reactive or feline-specific antibody will be required to further investigate EpCAM expression in normal and neoplastic feline tissue or for detecting CTCs in the blood of tumor-bearing cats.

Comparison of prevalence of Felis catus papillomavirus type 2 in squamous cell carcinomas in cats between Taiwan and Japan.

Felis catus papillomavirus (FcaPV), especially type 2 (FcaPV2) is considered as one of the causative agents in squamous cell carcinoma (SCC) in cats. However, our previous study detected FcaPV3 and FcaPV4, but not FcaPV2 in feline SCCs collected in Japan, suggesting that the prevalence of FcaPV2 in SCC may vary depending on geographic locations. To evaluate this hypothesis, two conventional PCR reactions targeting E1 and E7 genes were performed to detect FcaPV2 in feline SCC samples collected in Taiwan and Japan. While 46.9% (23/49) of feline SCC cases from Taiwan were PCR positive for FcaPV2, only 8.6% (3/35) cases from Japan were positive. Our result suggests that the prevalence of FcaPV2 in feline SCCs may depend on the region.

Computed Tomographic and Histopathological Characteristics of 13 Equine and 10 Feline Oral and Sinonasal Squamous Cell Carcinomas

Squamous cell carcinoma (SCC) is the most common equine sinonasal and feline oral tumour. This study aimed to describe the computed tomographic and histopathological characteristics of equine and feline SCC. Thirteen horses and 10 cats that had been histopathologically diagnosed with oral or sinonasal SCC and had undergone computed tomography (CT) of the head were retrospectively included in the study. CT characteristics of the mass and involved structures were noted. Histological examinations were evaluated according to a human malignancy grading system for oral SCC, which considered four grades of increasing aggressiveness. In horses, the masses were at the levels of the paranasal sinuses (n = 8), mandible (n = 3), tongue (n = 1), and nasal cavity (n = 1). In cats, the masses were at the levels of the maxilla (n = 4), mandible (n = 3), tongue (n = 1), and buccal region (n = 1) and were diffusely distributed (facial and cranial bones; n = 1). Masses in the equine paranasal sinuses showed only mild, solid/laminar, periosteal reactions with variable cortical destruction. However, maxillary lesions in cats showed severe cortical destruction and irregular, amorphous/pumice stone-like, periosteal reactions. CT revealed different SCC phenotypes that were unrelated to the histological grade. For morphologic parameters of the tumour cell population, a variability for the degree of keratinization and number of mitotic cells was noted in horses and cats. Concerning the tumour-host relationship a marked, extensive and deep invasion into the bone in the majority of horses and cats was seen. Most cases in both the horses and cats were categorized as histological grade III (n = 8); four horses and one cat were categorized as grade IV, and one horse and one cat were categorized as grade II. In this study, we examined the diagnostic images and corresponding applied human histopathological grading of SCC to further elucidate the correlations between pathology and oral and sinonasal SCC imaging in horses and cats.

Expression levels of angiogenic growth factors in feline squamous cell carcinoma.

Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the skin in cats. Tumour angiogenesis is the pivotal event for tumour progression and metastasis. We assessed protein and gene expression of angiogenic growth factors including bFGF, VEGF-C, TGF-β, PDGF-A, PDGF-C and PDGFR-α that possibly contribute to the angiogenic phenotype of feline SCC (FSCC) and could, therefore, be a good target in the treatment of SCC. In the present study, a total of 27 FSCC cases were investigated. Tumour cases were histopathologically classified as well differentiated (10/27), moderately differentiated (5/27), and poorly differentiated (12/27). The expression levels of the growth factors were detected using immunohistochemistry and assessed semi-quantitatively. Growth factor expression levels were evaluated at different locations: in the oral region, in areas exposed to solar UV radiation including the ears, eyelids and nasal planum, and other miscellaneous locations. Our findings have revealed that FSCC arising from different anatomical sites of the body and showing differences in aggressiveness, metastasis, and prognosis may be angiogenesis dependent, and angiogenic key regulators could play a role in the development of FSCC.

In vitro antiproliferation activity of Typhonium flagelliforme leaves ethanol extract and its combination with canine interferons on several tumor-derived cell lines.

Background and Aim:Tumor disorder is one of the degenerative diseases that affected human and animals and recently is tend to increase significantly. The treatment of tumor diseases can be performed through surgical, chemotherapy, radiotherapy, biological substances, and herbs medicine. Typhonium flagelliforme leaves extract known to have an antiproliferation activity, while interferons (IFNs) one of the cytokines that first used as an antiviral agent was also known to have antitumor activity. Nowadays, the treatment of tumors using a traditional way, including the use of herbal substances, becomes popular. Some limitations of the antitumor activity due to resistant development of the cell to some substances were one of the problems on why the treatment of cancer was unsuccessful. This study aimed to elaborate the synergistic effect on the antiproliferation and anti-angiogenesis activities of the combinations between T. flagelliforme leaves ethanol extract and canine natural (natural canine IFN [nCaIFN]) and recombinant (recombinant canine IFN [rCaIFN]) IFNs on tumor-derived cell lines to find the new potential antitumor substances.Materials and Methods:The extraction of T. flagelliforme leaves was performed using the maceration method and followed by phytochemical screening assays. According to the result of LC50 by the brine shrimp lethality test, the dose used for T. flagelliforme extract was 120 ppm while the dose of IFNs was 102 U/ml. The tumor-derived cell lines (canine squamous cell carcinoma [CSCC], canine mammary gland benign mixed tumor/MCM-IPB-B3, and feline squamous cell carcinoma [FSCC]) and normal rabbit endothelial cells were cultured and maintained on Dulbecco’s Modified Eagle’s Medium DMEM/Ham-F12 medium supplemented with 10% fetal calf serum, antibiotic, and antifungal. The antiproliferation activity was assayed by calculated the total cell number after treated with the tested substances. The antiangiogenesis assay was performed using in vitro method on rabbit normal endothelial cells and in ovo using chicken chorioallantoic membrane (CAM).Results:The phytochemical screening test of the T. flagelliforme leaves ethanol extract indicated that the compound consisted of flavonoid, steroid, and tannin. The antiproliferation activity was increased in the combination of substances compared to the single exposure of each substance on all tested tumor-derived cell lines. There was no significantly different on the antiproliferation activity between a combination of T. flagelliforme with nCaIFN or rCaIFN in every single tested cell lines, but the comparison of this activity among the three tumor-derived cell lines seem that the antiproliferation activity is more effective on CSCC cell lines compared to the canine mammary gland benign mixed tumor and FSCC cell lines. A similar pattern of synergistic effect was also detected on the anti-angiogenesis activity in vitro using rabbit endothelial cells as well as in ovo assays. The most effective of the in vitro and in ovo anti-angiogenesis activity was observed on the combination substances between T. flagelliforme extract and rCaIFN compared to other treatments.Conclusion:There was a synergistic effect on the antiproliferation and antiangiogenesis activities of the combination between T. flagelliforme and canine IFNs (natural and recombinant) and this result could be developed as another alternative on the cancer treatments.

Felis catus papillomavirus type-2 E6 binds to E6AP, promotes E6AP/p53 binding and enhances p53 proteasomal degradation

E6 from high risk human papillomaviruses (HR HPVs) promotes ubiquitination and degradation of p53 tumour suppressor by mediating its binding to ubiquitin ligase E6AP in a ternary complex, contributing to cell transformation in cervical cancer. We have previously shown that Felis catus papillomavirus type −2 (FcaPV-2) E6 is expressed in feline squamous cell carcinoma (SCC) and displays the ability to bind p53 and decrease its protein levels in transfected CRFK cells. However, the mechanism of p53 downregulation has not yet been characterized. Here we show that FcaPV-2 E6 bound to E6AP, which in turn was bound by p53 exclusively in cells expressing the viral oncoprotein (CRFKE6). Furthermore, p53 was highly poly-ubiquitinated and underwent accumulation upon E6AP gene knockdown in CRFKE6. Half-life experiments and proteasome inhibition treatments indicated that down-regulation of p53 protein in CRFKE6 was due to accelerated proteasomal degradation. E6AP/p53 binding was also demonstrated in two feline SCC cell lines expressing FcaPV-2 E6, where p53 protein levels and poly-ubiquitination degree were proportional to E6 mRNA levels. The data obtained in both artificial and spontaneous in vitro models suggest that FcaPV-2 E6 degrades p53 through a molecular mechanism similar to HR HPVs, possibly contributing to the development of feline SCC.

Surgical treatment using CO2 laser of feline squamous cell carcinoma of nasal planum in 6 cats

Surgical treatment using CO2 laser of feline squamous cell carcinoma of nasal planum in 6 cats

Epitope Mapping of Monoclonal Antibody PMab-52 Against Cat Podoplanin.

The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer, and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN) monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis and successfully identifies cPDPN in feline squamous cell carcinomas. However, the specific binding epitope of cPDPN for PMab-52 remains unelucidated. In this study, a series of deletion or point mutants of cPDPN were utilized for investigating the binding epitopes of PMab-52 using flow cytometry and Western blotting. The findings of this study revealed that the critical epitopes of platelet aggregation-stimulating domain 4 (PLAG4) of cPDPN are responsible for the binding of PMab-52 to cPDPN.

Evaluation of long-term outcome and prognostic factors of feline squamous cell carcinomas treated with photodynamic therapy using liposomal phosphorylated meta-tetra(hydroxylphenyl)chlorine.

The aim of this study was to evaluate the efficacy, long-term outcome and prognostic factors of feline squamous cell carcinoma (SCC) treated with photodynamic therapy (PDT). Cats with histologically verified SCC of the head and neck received an intravenous injection of liposomal phosphorylated meta-tetra(hydroxylphenyl)chlorine (mTHPC) and 4 h later 652 nm light was delivered by a diode laser. One group received ⩽10 J/cm2, the other 20 J/cm2. Tumour response and duration were analysed with stage, tumour diameter, location and treatment intensity as prognostic factors. In total, 63 lesions in 38 cats underwent treatment with ⩽10 J/cm2 (n = 22) and 20 J/cm2 (n = 41). Overall response rate was 84% (complete remission 61%, partial remission 22%) with a mean progression-free interval of 35 months (median not reached) and a median overall survival time of 40 months (95% confidence interval 33-47). With regard to tumour stage, invasiveness yielded a highly significant worse outcome ( P <0.017). All patients with invasive tumours showed progression at less than 6 months. Larger lesions were associated with inferior control and treatment intensity, and tumour location did not influence response and duration. PDT using a systemic photosensitiser leads to excellent long-term tumour control in the majority of cats. However, invasive and large tumours had a clearly inferior outcome, even if treated with the higher-dose intensity. This suggests that advanced lesions are not indications for PDT.

Localization of Felis catus Papillomavirus Type 2 E6 and E7 RNA in Feline Cutaneous Squamous Cell Carcinoma.

Findings from polymerase chain reaction-based methods have suggested a role of Felis catus papillomavirus 2 (FcaPV-2) in the development of feline cutaneous squamous cell carcinoma (SCC). However, because polymerase chain reaction cannot localize deoxyribonucleic acid or ribonucleic acid within the lesion, it is difficult to differentiate a coincidental FcaPV-2 infection and a causative association. Given that a key event in the pathogenesis of human papillomavirus-induced cancer is the expression of viral E6 and E7 oncogenes, localization of FcaPV-2 E6 and E7 transcription within neoplastic cells in feline SCCs would support a causative role for this papillomavirus. Therefore, RNAscope in situ hybridization was used to localize FcaPV-2 E6 and E7 transcripts in 18 formalin-fixed paraffin-embedded samples of cutaneous SCC. Positive signals were present within 5 of 9 samples (56%) from ultraviolet-protected sites and 0 of 9 samples from ultraviolet-exposed sites. In the 4 in situ hybridization-positive samples that contained adjacent hyperplastic skin, hybridization patterns in these regions were characterized by intense nuclear signals within the superficial epidermis and punctate signals within the basal epithelial layers. However, within the 5 SCCs, punctate signals were present within all layers of the epidermis, with progressive loss of intense nuclear signals within the superficial epidermis. This hybridization pattern is consistent with unregulated E6 and E7 transcription and decreased viral replication and is similar to the pattern observed in human papillomavirus-induced cancers as they progress from hyperplastic lesions containing productive infections to nonproductive neoplasms. These findings support a causative role for FcaPV-2 in the pathogenesis of feline SCC.

Detection of felis catus papillomavirus type 3 and 4 DNA from squamous cell carcinoma cases of cats in Japan.

Association of felis catus papillomaviruses (FcaPVs) with feline squamous cell carcinoma (SCC) has been reported worldwide, while there is limited information about FcaPVs in Asia. In this study, 21 feline SCC biopsy samples from cats in Japan were analyzed by PCR with PV consensus primers and type-specific primers for FcaPV type 2 (FcaPV-2), FcaPV-3 and FcaPV-4 designed in this study. Sequence analysis revealed that one sample was FcaPV-3, and two were FcaPV-4. In both FcaPV-4 positive samples, 334th tryptophan in L1 ORF was deleted compared with the reference sequence. Moreover, immunohistochemistry showed that p16 protein was positive in both FcaPV-4 detected samples. This study would contribute to the molecular epidemiological and pathological understanding of FcaPV in Japan.

Expression of Cat Podoplanin in Feline Squamous Cell Carcinomas

Oral squamous cell carcinoma is an aggressive tumor in cats; however, molecular-targeted therapies against this tumor, including antibody therapy, have not been developed. Sensitive and specific monoclonal antibodies (mAbs) against highly expressed membrane proteins are needed to develop antibody therapies. Podoplanin, a type I transmembrane glycoprotein, is expressed in many human malignant tumors, including brain tumor, esophageal cancer, lung cancer, mesothelioma, and oral cancer. Podoplanin binds to C-type lectin-like receptor-2 (CLEC-2) and activates platelet aggregation, which is involved in cancer metastasis. Until now, we have established several mAbs against podoplanin in humans, mice, rats, rabbits, dogs, cattle, and cats. We have reported podoplanin expression in canine melanoma and squamous cell carcinomas using an anti-dog podoplanin mAb PMab-38. In this study, we investigated podoplanin expression in 40 feline squamous cell carcinomas (14 cases of mouth floor, 13 of skin, 9 of ear, and 4 of tongue) by immunohistochemical analysis using an anti-cat podoplanin mAb PMab-52, which we recently developed by cell-based immunization and screening (CBIS) method. Of the total 40 cases, 38 (95%) showed positive staining for PMab-52. In particular, 12 cases (30%) showed a strong membrane-staining pattern of squamous cell carcinoma cells. PMab-52 can be useful for antibody therapy against feline podoplanin-expressing squamous cell carcinomas.

Felis catus papillomavirus type-2 but not type-1 is detectable and transcriptionally active in the blood of healthy cats.

Papillomaviruses (PVs) are small DNA viruses that induce benign and/or malignant epithelial tumours in different species, including the domestic cat (Felis catus). To date, five F.catus papillomavirus genotypes have been identified (FcaPV-1 to FcaPV-5). FcaPV-1 is associated with skin and oral benign lesions, while FcaPV-2 infection is widely associated with feline squamous cell carcinomas. Several human and animal PVs have been found in body fluids such as peripheral blood; however, the presence of FcaPVs in non-epithelial tissues has not previously been investigated. The aim of this study was to assess the presence and gene expression of FcaPV-1 and FcaPV-2 in the blood of healthy cats. We detected FcaPV-2 DNA in 26 of 103 (25%) blood samples. Importantly, FcaPV-2L1, E2, E6 and E7 genes were found to be expressed in 3 (25%), 11 (92%), 6 (50%) and 5 (42%) of the samples available for mRNA analysis, respectively. FcaPV-1 was not detected in any of the blood samples analysed here. The data obtained in this work suggest active and eventually productive infection of FcaPV-2 in the blood of healthy cats, implying a possible role in intra-individual spreading as well as in vertical and horizontal transmission.

Evaluation of protein kinase CK2 as a therapeutic target for squamous cell carcinoma of cats.

OBJECTIVE To investigate protein kinase CK2 (CK2) expression in squamous cell carcinoma (SCC) of cats and to examine effects of CK2 downregulation on in vitro apoptosis and viability in SCC. SAMPLE Biopsy specimens of oral mucosa and testis and blood samples from clinically normal cats, biopsy specimens of oral SCC from cats, and feline SCC (SCCF1) and mammary gland carcinoma (K12) cell lines. PROCEDURES Immunohistochemical labeling for CK2α was performed on biopsy specimens. Sequences of the CK2α subunit gene and CK2α' subunit gene in feline blood and feline cancer cell lines were determined by use of PCR and reverse-transcription PCR assays followed by direct Sanger sequencing. Specific small interfering RNAs (siRNAs) were developed for feline CK2α and CK2α'. The SCCF1 cells were treated with siRNA and assessed 72 hours later for CK2α and CK2α' expression and markers of apoptosis (via western blot analysis) and for viability (via 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium assays). RESULTS CK2α was expressed in all feline oral mucosa samples and 7 of 8 oral SCC samples. Expression of CK2α and CK2α' was successfully downregulated in SCCF1 cells by use of siRNAs, which resulted in decreased viability and induction of apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE In this study, CK2 appeared to be a promising therapeutic target for SCCs of cats. A possible treatment strategy for SCCs of cats would be RNA interference that targets CK2.

PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry.

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

Felis catus papillomavirus type 2 E6 oncogene enhances mitogen-activated protein kinases and Akt activation but not EGFR expression in an in vitro feline model of viral pathogenesis

A possible causative role of Felis catus papillomavirus type 2 (FcaPV2) in the development of feline oral and cutaneous squamous cell carcinomas (SCC) has been recently suggested by demonstrating viral gene expression in vivo and transforming properties by its putative oncogenes E6 and E7 in vitro. The activated molecules MEK (pMEK), ERK (pERK) and Akt (pAkt) are signaling transduction effectors regulating cell proliferation and inhibition of apoptosis, which are critical steps towards tumour formation. Here, we show by Western blotting (WB) that expression of FcaPV2 E6 in feline epithelial cells enhances pMEK, pERK and pAkt levels compared to control cells. Additionally, we demonstrated by real-time quantitative PCR on epidermal growth factor receptor (EGFR) transcripts and WB that activation of these signaling routes is independent from EGFR differential gene expression, total protein levels or phosphorylation, unlike in human papillomavirus associated tumours. This study contributes to define the molecular scenario underlying FcaPV2-triggered pathogenesis of feline SCC.