Margined Horn-Shaped Air Chamber for Body-Conduction Microphone.

The sound amplification ratios of sealed air chambers with different shapes were quantitatively compared to design a body-conduction microphone to measure animal scratching sounds. Recently, quantitative monitoring of scratching intensity in dogs has been required. We have already developed a collar with a body-conduction microphone to measure body-conducted scratching sounds. However, the air chamber, one of the components of the body-conduction microphone, has not been appropriately designed. This study compared the amplification ratios of air chambers with different shapes through numerical analysis and experiments. According to the results, the horn-shaped air chamber achieved the highest amplification performance, at least for sound frequencies below 3 kHz. The simulated amplification ratio of the horn-shaped air chamber with a 1 mm height and a 15 mm diameter was 52.5 dB. The deformation of the bottom of the air chamber affected the amplification ratio. Adjusting the margin of the margined horn shape could maintain its amplification ratio at any pressing force. The simulated and experimental amplification ratios of the margined horn-shaped air chamber were 53.4 dB and 19.4 dB, respectively.

BRAFV595E Mutation Associates CCL17 Expression and Regulatory T Cell Recruitment in Urothelial Carcinoma of Dogs.

Regulatory T cells may serve as targets in cancer immunotherapy. A previous study showed that the chemokine CCL17 and the receptor CCR4 play roles in regulatory T cell recruitment in canine urothelial carcinoma. In this article, we show that the BRAFV595E mutation is associated with tumor-produced CCL17 and regulatory T cell infiltration in dogs with urothelial carcinoma. In comparison with healthy dogs, dogs with urothelial carcinoma showed increased CCL17 mRNA expression in the bladder and elevated CCL17 protein concentration in urine. Immunohistochemistry showed increased levels of Foxp3+ regulatory T cells in the tumor tissues of urothelial carcinoma. The density of Foxp3+ regulatory T cells was positively correlated with CCL17 concentration in urine, indicating that CCL17 is involved in regulatory T cell recruitment. Moreover, tumor-infiltrating regulatory T cells and urine CCL17 concentration were associated with poor prognosis in dogs with urothelial carcinoma. The number of tumor-infiltrating regulatory T cells, CCL17 mRNA expression, and urine CCL17 concentration in cases with BRAFV595E mutation were higher than those in cases with wild-type BRAF. In vitro, high CCL17 production was detected in a canine urothelial carcinoma cell line with BRAFV595E mutation but not in an urothelial carcinoma cell line with wild-type BRAF. Dabrafenib, a BRAF inhibitor, decreased CCL17 production in the cell line with BRAFV595E mutation. These results suggest that BRAFV595E mutation induced CCL17 production and contributed to regulatory T cell recruitment in canine urothelial carcinoma.

Phosphatidic acid regulates subcellular distribution of RA-GEFs critical for chemokine-dependent migration.

Integrin activation by Rap1-GTP is pivotal for lymphocyte trafficking. In this study, we show the phosphatidic acid (PA)-dependent membrane distribution of RA-GEF-1 and -2 (also known as Rapgef2 and 6), which are guanine nucleotide exchange factors for Rap1, plays important roles in lymphocyte migration. RA-GEF-1 associates with PA through 919-967 aa within CDC25 homology domain, and the deletion of this region of RA-GEF-1 inhibits chemokine-dependent migration. Chemokine stimulation induces temporal production of PA on the plasma membrane, which is not necessary for Rap1 activation, but the translocation of RA-GEFs. Thus, chemokine-dependent generation of PA is critical for lymphocyte migration through membrane localization of RA-GEFs.

Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs.

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3+ Tregs and CCR4+ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4+ cells was associated with that of Foxp3+ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3+ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.

Myeloid derived suppressor cells in peripheral blood can be a prognostic factor in canine transitional cell carcinoma.

Myeloid-derived suppressor cells (MDSCs) are immature cells with immunosuppressive properties found in the tumor microenvironment. MDSCs are divided into two major subsets: polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). Both MDSC subsets contribute to the creation of an immunosuppressive environment for tumor progression. In humans, patients with high levels of MDSCs show worse outcomes for several types of cancers. However, the association between MDSCs and clinical features has rarely been investigated in canine studies. In the present study, we measured the proportion of PMN-MDSCs and M-MDSCs in the peripheral blood and tumor tissue of dogs with hepatocellular carcinoma (HCC), prostate cancer (PC), transitional cell carcinoma (TCC), lymphoma, and pulmonary adenocarcinoma. Additionally, we examined immunosuppressive ability of PMN-MDSCs and M-MDSCs in peripheral blood mononuclear cells of TCC case on CD4+, CD8+ and interferon-γ+ cells and investigated the relationships of MDSCs with clinical features and outcomes. PMN-MDSCs increased in HCC, PC, TCC, and lymphoma. In contrast, M-MDSCs increased in the TCC. Both PMN-MDSCs and M-MDSCs exhibited immunosuppressive effects on CD8+, CD4+ and interferon-γ+ cells. In dogs with TCC, lymph node metastasis was associated with high level of PMN-MDSCs but not with M-MDSCs. High levels of both PMN-MDSCs and M-MDSCs were related to advanced tumor stage. Kaplan-Meier analysis revealed that high levels of both PMN-MDSCs and M-MDSCs were significantly associated with shorter overall survival. In addition, the Cox proportional hazard regression model showed that M-MDSCs and the tumor stage were independent prognostic factors for TCC. These results suggest that PMN-MDSCs and M-MDSCs may be involved in tumor progression and could be prognostic factors and promising therapeutic targets in dogs with TCC.

Effect of diazoxide on a cat with insulinoma.

Case summary: The patient was a castrated male American Shorthair cat, approximately 14 years old, weighing 3.4 kg. The patient had chronic kidney disease (CKD) (International Renal Interest Society stages 3-4) as an underlying disease. The cat was examined at a hospital for intermittent lethargy and seizures. Hypoglycaemia was repeatedly observed, and the insulin level was 1.78 ng/ml (reference interval 0.27-0.69) when the blood glucose was 49 mg/dl. Although the cat was tentatively diagnosed with insulinoma, surgery was not recommended because of the severe CKD. Although frequent feeding and prednisolone treatment were initially attempted, blood glucose decreased to 24-42 mg/dl. Diazoxide was additionally prescribed at a dose of 5.2 mg/kg q12h. The cat's clinical signs improved, and the blood glucose was in the range of 75-103 mg/dl during the first 2 months. It was maintained at >50 mg/dl until the patient died of renal failure 161 days after the start of diazoxide treatment. With regard to adverse events, vomiting once every 2-3 days without weight loss and non-regenerative anaemia were observed, which might have been at least partially caused by diazoxide treatment. An insulinoma was definitively diagnosed via pathological autopsy. Relevance and novel information: This is the first reported case of long-term treatment with diazoxide in a cat with insulinoma. Since it was effective in situations where conventional therapies were unsuccessful, diazoxide could be useful as a new therapeutic option for cats with insulinoma. Since adverse events, such as progression of vomiting frequency and non-regenerative anaemia, were observed, careful monitoring was required during administration.

Anti-tumour effects of lapatinib on HER2-positive canine prostatic carcinoma cell lines.

Background: Canine prostatic carcinoma (cPC) is a urogenital tumour with a poor prognosis, for which no effective treatment has been established. Recently, it has been shown that human epidermal growth factor receptor type 2 (HER2) is overexpressed in cPC cells; however, the efficacy of HER2-targeted therapy remains unclear. Aim: Investigate the anti-tumour effect of lapatinib on HER2-positive cPC cell lines. Methods: Two cell lines (muPC and bePC) were established from two dogs with cPC and the effects of lapatinib treatment on cell proliferation, apoptosis, and HER2 downstream signalling were investigated. Furthermore, muPC was used to generate tumour-bearing mice, and the anti-tumour effects of lapatinib were examined in vivo. Results: Lapatinib treatment inhibited the proliferation and phosphorylation of Erk1/2 and Akt, which are downstream signals of HER2. Furthermore, the TUNEL assay showed that lapatinib induced apoptosis in both cell lines. The muPC-engrafted nude mouse model showed that lapatinib significantly inhibited tumour growth and increased the area of necrotic tumour tissue compared to the vehicle-treated groups. Conclusion: Lapatinib exerts anti-tumour effects on cPC cells by inhibiting HER-2 signalling.

Elucidation of prognostic factors in the acute phase of feline severe fever with thrombocytopenia syndrome virus infection.

Severe fever with thrombocytopenia syndrome (SFTS) is a potentially fatal tick-borne zoonotic disease, endemic to Asian regions, including western Japan. Cats appear to suffer a particularly severe form of the disease; however, feline SFTS is not clinically well characterized. Accordingly, in this study, we investigated the associations of, demographic, hematological and biochemical, immunological, and virological parameters with clinical outcome (fatal cases vs. survivors) in SFTSV-positive cats. Viral genomic analysis was also performed. Viral load in blood, total bilirubin, creatine phosphokinase, serum amyloid A, interleukin-6, tumor necrotic factor-α, and virus-specific IgM and IgG differed significantly between survivors and fatal cases, and thus may have utility as prognosticators. Furthermore, survivor profiling revealed high-level of viremia with multiple parameters (white blood cells, platelet, total bilirubin, glucose, and serum amyloid A) beyond the reference range in the 7-day acute phase, and signs of clinical recovery in the post-acute phase (parameters returning to, or tending toward, the reference range). However, SFTSV was still detectable from some survived cats even 14 days after onset of disease, indicating the risk of infection posed by close-contact exposure may persist through the post-acute phase. This study provides useful information for prognostic assessments of acute feline SFTS, and may contribute to early treatment plans for cats with SFTS. Our findings also alert pet owners and animal health professionals to the need for prolonged vigilance against animal-to-human transmission when handling cats that have been diagnosed with SFTS.

Detection of feline morbillivirus in cats with symptoms of acute febrile infection.

Feline morbillivirus (FeMV) was identified for the first time in cats in 2012 in Hong Kong. Although its association with chronic kidney disease in cats has attracted the attention of researchers, its clinical significance as an acute infection has not been reported. Previously, we reported FeMV detection using next-generation sequence-based comprehensive genomic analysis of plasma samples from cats with suspected acute febrile infections. Here, we conducted an epidemiological survey to detect FeMV by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using blood samples from cats in Japan. FeMV was detected in 32/102 blood samples (31.4%) from cats with suspected acute viral infections. Most of the FeMV-positive cats had clinical findings consistent with acute viral infections, including fever, leukopenia, thrombocytopenia and jaundice. No FeMV was detected in healthy cats or clinically ill cats that visited veterinary hospitals. Phylogenetic analysis classified FeMV L genes into various FeMV subtypes. We also necropsied a FeMV-positive cat that died of a suspected acute infection. On necropsy, FeMV was detected in systemic organs, including the kidneys, lymph nodes and spleen by qRT-PCR and immunohistochemical staining. These results suggest that FeMV infections may cause acute symptomatic febrile infections in cats. A limitation of this study was that the involvement of other pathogens that cause febrile illnesses could not be ruled out and this prevented a definitive conclusion that FeMV causes febrile disease in infected cats. Further studies that include experimental infections are warranted to determine the pathogenicity of FeMV in cats.

Protective role of protease-activated receptor-2 in anaphylaxis model mice.

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

8-Hydroxy-2'-deoxyguanosine and malondialdehyde in plasma and their association with disease severity in 20 cats with chronic kidney disease.

OBJECTIVES: Oxidative stress is associated with the development and progression of chronic kidney disease (CKD) in humans. The aim of this study was to evaluate the concentrations of oxidative stress markers, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and malondialdehyde (MDA), in the plasma and urine of cats with different stages of CKD. METHODS: Plasma and urine samples were collected from cats with CKD that were referred to the Veterinary Medical Center of the University of Tokyo between April 2019 and October 2022. Plasma and urine samples were collected from healthy cats (n = 6 at most), cats with stage 2 CKD (n = 8) and stage 3-4 CKD (n = 12), and cats with idiopathic cystitis (disease control, n = 5). Plasma and urine concentrations of 8-OHdG and MDA were measured using ELISA and thiobarbituric acid reactive substances assay kits, respectively. RESULTS: The median plasma 8-OHdG concentrations were 0.156 ng/ml (<0.125-0.210 ng/ml) in the healthy group, <0.125 ng/ml (range <0.125 ng/ml) in the idiopathic cystitis group, 0.246 ng/ml (range 0.170-0.403 ng/ml) in cats with stage 2 CKD and 0.433 ng/ml (range 0.209-1.052 ng/ml) in cats with stage 3-4 CKD. Concentrations in stage 3-4 CKD were significantly higher than those in the healthy and disease control groups. Plasma MDA concentrations were low in the healthy and disease control groups and significantly higher in cats with stage 3-4 CKD. In every cat with CKD, plasma 8-OHdG and MDA concentrations were positively correlated with plasma creatinine concentrations (8-OHdG, rs = 0.68; MDA, rs = 0.67). Urinary 8-OHdG/urinary creatinine (u-CRE) and urinary MDA/u-CRE levels did not differ significantly between the groups; however, it was difficult to evaluate them because of the small sample size. CONCLUSIONS AND RELEVANCE: This report shows that plasma 8-OHdG and MDA concentrations increase with the severity of feline CKD. These markers may be useful for assessing oxidative stress in cats with CKD.

Effects of high-dose docosahexaenoic acid supplementation as an add-on therapy for canine idiopathic epilepsy: A pilot study.

Background: The anti-epileptic effects of docosahexaenoic acid (DHA) in dogs and humans remain controversial. The dosage and efficacy of DHA were various in the previous reports. Aim: The effects of high-dose DHA supplementation as add-on therapy for idiopathic epilepsy in dogs were evaluated. Methods: An open-label clinical trial was designed in this pilot study. Six dogs (median age: 6 years) with idiopathic epilepsy were included. All the patients were diagnosed with idiopathic epilepsy using magnetic MRI and cerebrospinal fluid examination (median: 2.0 years before the trial). They had 5-45 seizures and/or auras (median: 9.0) in the month before starting DHA supplementation. DHA was adjunctively administered at doses of 69-166 mg/kg/day without changing other prescriptions. Results: Four of the six patients completed the 6-month observation period. All the patients showed a decrease in seizure frequency of 50% or more within 2-3 months after the start of the administration, and three patients decreased to a frequency of 0-1 per month after 5-6 months. No clear adverse events were observed in the general condition or blood test results in any patients. Conclusion: Although the sample size was small and the study was not a randomized controlled trial, the data suggest that add-on supplementation of DHA could be useful in reducing the frequency of seizures in canine idiopathic epilepsy.

Detection of domestic cat hepadnavirus by next-generation sequencing and epidemiological survey in Japan.

The novel domestic cat hepadnavirus (DCH), a member of the Hepadnaviridae, was first detected in Australia and has recently been identified in more countries. In this study, we explored the DCH genome using next-generation sequencing of a plasma sample from a cat with a fever of unknown cause. Nucleotide sequence analysis showed the virus to be relatively genetically distant from the first reported DCH in Australia, showing 89% homology. Then we conducted an epidemiological survey by PCR of plasma samples collected from 203 cats that visited a veterinary hospital for diagnosis and treatment. Two of the 203 surveyed cats a were positive for DCH. One of the two positive cases had elevated liver enzymes of unknown etiology, and the other had hepatocellular adenoma. Our study indicated that DCH infection was observed in domestic cats in the Tokyo area of Japan as well as other reported areas in the world. Further investigations are needed to define the clinical importance of DCH.

Large-scale epidemiological study on feline autosomal dominant polycystic kidney disease and identification of novel PKD1 gene variants.

OBJECTIVES: Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disease in cats. In most cases, the responsible abnormality is a nonsense single nucleotide polymorphism in exon 29 of the PKD1 gene (chrE3:g.42858112C>A, the conventional PKD1 variant). The aim of this study was to conduct a large-scale epidemiological study of ADPKD caused by the conventional PKD1 variant in Japan and to search for novel polymorphisms by targeted resequencing of the PKD1 using a next-generation sequencer. METHODS: A total of 1281 cats visiting the Veterinary Medical Center of the University of Tokyo were included in this study. DNA was extracted from the blood of each cat. We established a novel TaqMan real-time PCR genotyping assay for the conventional PKD1 variant, and all cases were examined for the presence of this variant. Targeted resequencing of all exons of the PKD1 was performed on the DNA of 23 cats with the conventional PKD1 variant, six cats diagnosed with cystic kidneys but without this variant, and 61 wild-type normal cats. RESULTS: Among the 1281 cats examined in this study, 23 (1.8%) harboured the conventional PKD1 variant. The odds of having the conventional PKD1 variant were significantly higher in Persian cats, Scottish Folds and Exotic Shorthairs than in the other breeds, although the number of cases in each breed was small. Furthermore, we identified four variants unique to cats with cystic kidneys that were not found in wild-type normal cats, all of which were in exon 15. In particular, two (chrE:g.42848725delC, pGly1641fs and chrE:g.42850283C>T, pArg2162Trp) were candidate variants. CONCLUSIONS AND RELEVANCE: This study revealed that the conventional PKD1 variant was prevalent in Scottish Fold, Persian and Exotic Shorthair breeds in Japan, and variants in exon 15 of PKD1, in addition to the conventional variant in exon 29, would be key factors in the pathogenesis of ADPKD in cats.

Clinical and Pathological Findings in Fatal Cases of Severe Fever With Thrombocytopenia Syndrome With High Viremia in Cats.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease caused by the SFTS virus (SFTSV). SFTSV causes severe symptoms both in humans and cats. In this study, we report the clinical and pathological findings of 4 fatal cases of cats with high SFTS viremia levels. These cats showed an acute onset of fever, leukopenia, thrombocytopenia, and increased serum amyloid A and pro-inflammatory cytokine levels. A high viral copy number was detected in the blood, oral swabs, rectal swabs, conjunctiva swabs, and urine. Histopathologically, necrotizing lymphadenitis, splenitis with lymphoblastoid cell proliferation, and hemophagocytosis were observed in all 4 cats. Immunohistochemistry revealed the presence of SFTSV antigen on lymphoblastoid B cells. SFTSV-RNA was detected in systemic tissues, including the brain. The present findings provide useful information for understanding the features of fatal SFTS in cats. To elucidate the mechanisms of severe progress of SFTS cats, as well as its role as a source of human infection, further research is needed.

Serum level of apoptosis inhibitor of macrophage in dogs with histiocytic sarcoma and its association with the disease.

Histiocytic sarcoma (HS) is a rare neoplasm of macrophages or dendritic cells with a poor prognosis in dogs. As the apoptosis inhibitor of macrophage (AIM) is characteristically expressed in canine macrophages, we hypothesised that AIM is involved in the development or progression of HS in dogs. In this study, AIM expression in the tumour region and serum AIM levels in dogs with HS was assessed. Additionally, the effects of AIM overexpression on HS cell viability were investigated using a HS cell line that was selected from five validated HS cell lines. Immunohistochemistry showed that AIM expression was observed in the cytoplasm of the HS cells. CD36, a candidate AIM receptor, was also observed on the cell membrane of HS cells. When the serum AIM level was detected in 36 dogs with HS and 10 healthy dogs via western blot analysis, the AIM levels in the HS dogs were significantly higher than those in the controls. AIM mRNA expression in the 5 HS cell lines varied but was higher than that in the other tumour-derived lines. Among the five HS cell lines, DH82 originally had lower AIM and the highest CD36 expression. When AIM was overexpressed in DH82, therein cell growth speed and invasion, apoptosis inhibition and phagocytic activity were strongly upregulated. These data suggest that elevated intra-tumour expression of AIM could induce the progression of HS cells in dogs. Moreover, elevated serum AIM levels in dogs with HS could serve as a biomarker of HS.

Detection of Canine Urothelial Carcinoma Cells in Urine Using 5-Aminolevulinic Acid.

This study aimed to establish a method to detect canine urothelial carcinoma cells in urine using 5-aminolevulinic acid (5-ALA) and to evaluate its diagnostic accuracy. Urine samples were collected from 21 dogs diagnosed with urothelial carcinoma and three urothelial carcinoma cell lines were used. Urine samples obtained from seven healthy dogs were used as controls. Cells in the urine sediment, or urothelial carcinoma cell lines, were cultured with 5-ALA and then observed under a fluorescence microscope. Moreover, we examined the relationship between fluorescence intensity and the presence of metastasis as well as tumor invasion into the bladder wall in cases of urothelial carcinoma. Urine-derived cells from urothelial carcinoma and urothelial carcinoma cell lines showed clearer red fluorescence with the addition of 5-ALA compared to that exhibited by the cells from healthy dogs. The sensitivity and specificity of the diagnosis of urothelial carcinoma were 90% and 86%, respectively. Significant associations were found between fluorescence intensity and tumor metastasis and bladder wall invasion. This study showed that 5-ALA can be used to detect urothelial carcinoma cells in dogs with relatively high diagnostic accuracy. Further, the fluorescence intensity of tumor cells caused by 5-ALA correlated with the clinical condition of urothelial carcinoma cases, which suggested that 5-ALA could be used as a prognostic marker for canine urothelial carcinoma.

Sorafenib inhibits tumor cell growth and angiogenesis in canine transitional cell carcinoma.

Canine transitional cell carcinoma (cTCC) is the most common naturally occurring bladder cancer and accounts for 1-2% of canine tumors. The prognosis is poor due to the high rate of invasiveness and metastasis at diagnosis. Sorafenib is a multi-kinase inhibitor that targets rapidly accelerated fibrosarcoma (RAF), vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3, platelet-derived growth factor receptor-ß (PDGFR-ß), and KIT. In previous studies, a somatic mutation of B-rapidly accelerated fibrosarcoma (BRAF) and expressions of VEGFR-2 and PDGFR-ß were observed in over 80% of patients with cTCC. Therefore, in this study, we investigated the anti-tumor effects of sorafenib on cTCC. Five cTCC cell lines were used in the in vitro experiments. All five cTCC cell lines expressed VEGFR-2 and PDGFR-ß and sorafenib showed growth inhibitory effect on cTCC cell lines. Cell cycle arrest at the G0/G1 phase and subsequent apoptosis were observed following sorafenib treatment. In the in vivo experiments, cTCC (Sora) cells were subcutaneously injected into nude mice. Mice were orally administered with sorafenib (30 mg/kg daily) for 14 days. Sorafenib inhibited tumor growth compared to vehicle control. The necrotic area in the tumor tissues was increased in the sorafenib-treated group. Sorafenib also inhibited angiogenesis in the tumor microenvironment. Thus, sorafenib may be potential therapeutic agent for cTCC via its direct anti-tumor effect and inhibition of angiogenesis.

Lapatinib as first-line treatment for muscle-invasive urothelial carcinoma in dogs.

Epidermal growth factor receptors 1 and 2 (EGFR and HER2) are frequently overexpressed in various malignancies. Lapatinib is a dual tyrosine kinase inhibitor that inhibits both EGFR and HER2. Although a phase III trial failed to show the survival benefits of lapatinib treatment after first-line chemotherapy in patients with EGFR/HER2-positive metastatic urothelial carcinoma, the efficacy of lapatinib for untreated urothelial carcinoma is not well defined. Here, we describe the therapeutic efficacy of lapatinib as a first-line treatment in a canine model of muscle-invasive urothelial carcinoma. In this non-randomized clinical trial, we compared 44 dogs with naturally occurring urothelial carcinoma who received lapatinib and piroxicam, with 42 age-, sex-, and tumor stage-matched dogs that received piroxicam alone. Compared to the dogs treated with piroxicam alone, those administered the lapatinib/piroxicam treatment had a greater reduction in the size of the primary tumor and improved survival. Exploratory analyses showed that HER2 overexpression was associated with response and survival in dogs treated with lapatinib. Our study suggests that lapatinib showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use for untreated advanced urothelial carcinoma in dogs. The use of lapatinib as a first-line treatment may be investigated further in human patients with urothelial carcinoma.

Anti-CCR4 treatment depletes regulatory T cells and leads to clinical activity in a canine model of advanced prostate cancer.

BACKGROUND: Targeting regulatory T cell (Treg) infiltration is an emerging strategy for cancer immunotherapy. However, its efficacy in advanced prostate cancer remains unclear. Here, we showed the therapeutic efficacy of anti-Treg treatment in a canine model of advanced prostate cancer. METHODS: We used dogs with naturally occurring prostate cancer to study the molecular mechanism underlying Treg infiltration and the effect of anti-Treg treatment. Tumor-infiltrating Tregs was evaluated by immunohistochemistry, and the association with prognosis was examined in dogs with spontaneous prostate cancer. The molecular mechanism of Treg infiltration was explored by RNA sequencing and protein analyses. A non-randomized canine clinical trial was conducted to define the therapeutic potential of anti-Treg treatment for advanced prostate cancer. Human prostate cancer datasets were analyzed to compare gene expression in dogs and humans. RESULTS: Tumor-infiltrating Tregs were associated with poor prognosis in dogs bearing spontaneous prostate cancer. RNA sequencing and protein analyses showed a possible link between the CCL17-CCR4 pathway and the increase of tumor-infiltrating Tregs. Dogs with advanced prostate cancer responded to mogamulizumab, a monoclonal antibody targeting CCR4, with decreased circulating Tregs, improved survival, and low incidence of clinically relevant adverse events. Urinary CCL17 concentration and BRAFV595E mutation were independently predictive of the response to mogamulizumab. Analysis of a transcriptomic dataset of human prostate cancer showed that the CCL17-CCR4 axis correlated with Foxp3. In silico survival analyses revealed that high expression of CCL17 was associated with poor prognosis. Immunohistochemistry confirmed that tumor-infiltrating Tregs expressed CCR4 in human patients with prostate cancer. CONCLUSIONS: Anti-Treg treatment, through CCR4 blockade, may be a promising therapeutic approach for advanced prostate cancer in dogs and some population of human patients.