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Background: The use of adrenocorticotropic hormone stimulation test as method to monitor efficacy of trilostane treatment of hypercortisolism (HC) in dogs has been questioned. Objectives: To evaluate and compare 12 methods with which to monitor efficacy of trilostane treatment in dogs with HC. Animals: Forty-five client-owned dogs with HC treated with trilostane q12h. Methods: Prospective cross-sectional observational study. The dogs were categorized as well-controlled, undercontrolled, and unwell through a clinical score obtained from an owner questionnaire. The ability to correctly identify trilostane-treatment control of dogs with HC with the following variables was evaluated: before trilostane serum cortisol (prepill), before-ACTH serum cortisol, post-ACTH serum cortisol, plasma endogenous ACTH concentrations, prepill/eACTH ratio, serum haptoglobin (Hp) concentration, serum alanine aminotransferase (ALT), gamma-glutamyl transferase (γGT) and alkaline phosphatase activity, urine specific gravity, and urinary cortisol:creatinine ratio. Results: Ninety-four re-evaluations of 44 dogs were included; 5 re-evaluations of 5 unwell dogs were excluded. Haptoglobin was significantly associated with the clinical score (P

Canine Cushing's syndrome (hypercortisolism) can be caused by a pituitary tumor (pituitary-dependent hypercortisolism; PDH) or a cortisol-secreting adrenocortical tumor (csACT). For both cases, non-invasive biomarkers that could pre-operatively predict the risk of recurrence after surgery would greatly impact clinical decision making. The aim of this study was to determine whether circulating microRNAs (miRNAs) can be used as diagnostic (presence of PDH or csACT) and/or prognostic (disease recurrence, histological grade) non-invasive biomarkers for canine Cushing's syndrome. After a pilot study with 40 miRNAs in blood samples of healthy dogs (n = 3), dogs with PDH (n = 3) and dogs with a csACT (n = 4), we selected a total of 20 miRNAs for the definitive study. In the definitive study, these 20 miRNAs were analyzed in blood samples of healthy dogs (n = 6), dogs with PDH (n = 19, pre- and post-operative samples) and dogs with a csACT (n = 26, pre-operative samples). In dogs with PDH, six miRNAs (miR-122-5p, miR-126-5p, miR-141-3p, miR-222-3p, miR-375-3p and miR-483-3p) were differentially expressed compared to healthy dogs. Of one miRNA, miR-122-5p, the expression levels did not overlap between healthy dogs and dogs with PDH (p = 2.9x10−4), significantly decreased after hypophysectomy (p = 0.013), and were significantly higher (p = 0.017) in dogs with recurrence (n = 3) than in dogs without recurrence for at least one year after hypophysectomy (n = 7). In dogs with csACTs, two miRNAs (miR-483-3p and miR-223-3p) were differentially expressed compared to healthy dogs. Additionally, miR-141-3p was expressed significantly lower (p = 0.009) in dogs with csACTs that had a histopathological Utrecht score of ≥ 11 compared to those with a score of

Purpose: To find underlying mutations causing highly-activated Wnt activity in mammary tumor cell lines associated with rounded morphology indicative of stemness/EMT. Methods: Stemness of high Wnt cell lines was confirmed using qPCR on selected genes and microRNA profiling, followed by whole-exome sequencing of 3 high Wnt canine mammary tumor cell lines and 5 low/absent Wnt cell lines. Candidate genes were identified and their involvement in Wnt activity investigated using siRNA silencing. Results: The high Wnt cell lines had morphological and gene expression characteristics reminiscent of stemness. All individual cell lines had about 4000 mutations in the exome in comparison to the reference canine genome. The three high basal Wnt cell lines had 167 unique exome mutations. Seven of these mutations resulted in a SIFT score

The COVID-19 pandemic raised concerns that companion animals might be infected with, and could become a reservoir of, SARS-CoV-2. As cats are popular pets and susceptible to Coronavirus, we investigated the seroprevalence of SARS-CoV-2 antibodies in shelter cats housed in Dutch animal shelters during the COVID-19 pandemic. In this large-scale cross-sectional study, serum samples of shelter cats were collected during the second wave of human COVID-19 infections in The Netherlands. Seroprevalence was determined by using an indirect protein-based ELISA validated for cats, and a Virus Neutralization Test (VNT) as confirmation. To screen for feline SARS-CoV-2 shedding, oropharyngeal and rectal swabs of cats positive for ELISA and/or VNT were analyzed using PCR tests. In 28 Dutch animal shelters, 240 shelter cats were convenience sampled. Two of these cats (0.8%, CI 95%: 0.1–3.0%) were seropositive, as evidenced by the presence of SARS-CoV-2 neutralizing antibodies. The seropositive animals tested PCR negative for SARS-CoV-2. Based on the results of this study, it is unlikely that shelter cats could be a reservoir of SARS-CoV-2 or pose a (significant) risk to public health.

Pituitary tumours are common in dogs and are being increasingly recognized in cats. Pituitary tumours are usually classified as adenomas and should only be classified as carcinomas when there is evidence of metastatic spread of the tumour, which is rare. Despite the benign nature of most pituitary tumours, they can still compress or invade neighbouring tissues. Pituitary tumours can be functional (hormonally active) or non-functional (hormonally silent). The aim of this review was to provide an overview of the different pituitary tumour types in dogs and cats that have been reported in the literature. In dogs, the most common pituitary tumour type is the corticotroph adenoma, which can cause pituitary-dependent hypercortisolism. In cats, the most common pituitary tumour is the somatotroph adenoma, which can cause hypersomatotropism, and the second-most common is the corticotroph adenoma. A lactotroph adenoma has been described in one dog, while gonadotroph, thyrotroph and null cell adenomas have not been described in dogs or cats. Hormonally silent adenomas are likely underdiagnosed because they do not result in an endocrine syndrome. Tools used to classify pituitary tumours in humans, particularly immunohistochemistry for lineage-specific transcription factors, are likely to be useful to classify canine and feline pituitary tumours of unknown origin. Future studies are required to better understand the full range of pituitary adenoma pathology in dogs and cats and to determine whether certain adenoma subtypes behave more aggressively than others. Currently, the mechanisms that underlie pituitary tumorigenesis in dogs and cats are still largely unknown. A better understanding of the molecular background of these tumours could help to identify improved pituitary-targeted therapeutics.

Mesenchymal stromal cells (MSC) are used for cell-based treatment for canine osteoarthritis (OA). Compared with human MSCs, detailed information on the functional characterisation of canine MSCs is limited. In particular, the chondrogenic differentiation of canine adipose tissue-derived MSCs (cAT-MSCs) is challenging. In this study, we aimed to compare cAT-MSCs with bone marrow-derived MSCs (cBM-MSCs), focusing specifically on their in vitro chondrogenic potential, with or without bone morphogenetic proteins (BMP). cBM-MSCs and cAT-MSCs were characterised using flow cytometry and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The chondrogenic differentiation potential of all cMSC preparations in the presence of TGF-β1 alone or when supplemented with 10, 100, or 250 ng/mL BMP-2 or BMP-6 was investigated using RT-qPCR, and biochemical, histochemical and immunohistological analyses. Both cBM-MSCs and cAT-MSCs expressed the surface markers CD90, CD73, and CD29, and were negative for CD45 and CD34, although the expression of CD73 and CD271 varied with donor and tissue origin. Interestingly, expression of ACAN and SOX9 was higher in cBM-MSCs than cAT-MSCs. In contrast with cBM-MSCs, cAT-MSCs could not differentiate toward the chondrogenic lineage without BMP-2/-6, and their in vitro chondrogenesis was inferior to cBM-MSCs with BMP-2/-6. Thus, cAT-MSCs have lower in vitro chondrogenic capacity than cBM-MSC under the studied culture conditions with 10, 100, or 250 ng/mL BMP-2 or BMP-6. Therefore, further characterisation is necessary to explore the potential of cAT-MSCs for cell-based OA treatments.

Background Successful treatment of cats with hypersomatotropism by transsphenoidal hypophysectomy is described in small numbers of cats. Objectives To describe the endocrine profile, survival, and remission rates of hypersomatotropism and diabetes mellitus in a cohort of cats with hypersomatotropism that underwent hypophysectomy between 2008 and 2020. Animals Twenty-five client-owned cats with spontaneous hypersomatotropism. Methods Retrospective study. Diagnosis of hypersomatotropism was based on clinical signs, plasma insulin-like growth factor-1 (IGF-1) concentration, and imaging of the pituitary gland. Growth hormone (GH) and IGF-1 concentrations were measured repeatedly after surgery. Survival times were calculated based on follow-up information from owners and referring veterinarians. Results Median postoperative hospital stay was 7 days (range, 3-18 days). One cat died within 4 weeks of surgery. Median plasma GH concentration decreased significantly from 51.0 ng/mL (range, 5.0-101.0 ng/mL) before surgery to 3.8 ng/mL (range, 0.6-13.0 ng/mL) at 5 hours after surgery. Remission of hypersomatotropism, defined as normalization of plasma IGF-1 concentration, occurred in 23/24 cats (median, 34 ng/mL; range, 14-240 ng/mL) and 22/24 cats entered diabetic remission. Median survival time was 1347 days (95% confidence interval, 900-1794 days; range, 11-3180 days) and the overall 1-, 2-, and 3-year all-cause survival rates were 76%, 76%, and 52%, respectively. Conclusions and clinical importance This study shows the beneficial outcome of hypophysectomy in cats with hypersomatotropism, marked by low death rate and a high percentage of diabetic remission and definitive cure.

Objectives The aims of this study were to validate a commercially available chemiluminescent assay for measurement of feline plasma adrenocorticotropic hormone concentration (ACTH), to determine the normal reference interval (RI) of plasma ACTH in healthy cats, to assess plasma ACTH in cats with naturally occurring hypercortisolism (HC), primary hypoadrenocorticism (PH) and other diseases (OD), and to evaluate the effect of aprotinin on plasma ACTH degradation. Methods Forty healthy cats, 10 with HC, 11 with PH and 30 with OD, were included. The chemiluminescent enzyme immunometric assay was evaluated by measurement of intra-assay precision, interassay precision and linearity. The RI for plasma ACTH in healthy cats was established using robust methods. Plasma ACTH of samples collected with and without aprotinin, stored at 4°C and assayed over a 6-day period, was measured. Results The intra-assay coefficients of variance (CVs) ranged from 2.7% to 4.3% and interassay CVs from 3.3% to 10.7%. Dilution studies showed excellent accuracy (R2 >0.99). The RI for plasma ACTH in healthy cats was 32–370 pg/ml. Plasma ACTH was not significantly different between healthy cats and the OD group. Cats with pituitary-dependent hypercortisolism (PDH) and PH had significantly higher plasma ACTH than the other groups. Plasma ACTH did not show significant differences when samples collected with and without aprotinin were compared. Conclusions and relevance The Immulite chemiluminescent assay is a valid technique for measuring plasma ACTH in cats and the RI of plasma ACTH is quite wide. Owing to the low overlap between healthy or OD cats and cats with HC or PH, the measurement of plasma ACTH appears to be useful and should be included in the diagnostic work-up when HC or PH are suspected. Furthermore, the measurement of plasma ACTH may be an accurate test for differentiating PDH from adrenal-dependent hypercortisolism.

BACKGROUND Information on scintigraphy findings in dogs with thyroid neoplasia is scarce. The use of single-photon emission computed tomography (SPECT) could improve detection of metastatic disease. HYPOTHESIS/OBJECTIVES To describe planar and SPECT imaging findings in dogs with thyroid tumors, and to compare SPECT and thoracic radiography for metastasis detection. ANIMALS Sixty-eight dogs with thyroid neoplasia. METHODS Retrospective study, search of medical records for dogs with thyroid neoplasia (2008-2018). RESULTS Thyroid scintigraphy was available from 68 dogs, of which 6 presented after surgical resection. Radionuclide uptake was increased in 56% of dogs, decreased in 24%, and comparable to that of the salivary glands in 13%. The remainder had multiple masses with variable uptake. A homogeneous uptake pattern was present in 16% and a heterogeneous uptake pattern in 73%. In 11% (all dogs with multiple masses), various uptake patterns were present. Thyroid tumors were well delineated in 55%. There was a significant association between hormone status and uptake pattern (P = .009), with a heterogeneous uptake pattern in the majority of euthyroid dogs, and hormone status and tumor circumscription (P = .003), with well-circumscribed margins in the majority of hypothyroid and hyperthyroid dogs. Thoracic SPECT imaging was available in 39 dogs and identified metastatic lesions in 15 dogs. Thoracic radiographs were performed in 14 of these dogs, and detected metastases in 3 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE SPECT imaging is a viable imaging technique to screen for thoracic metastasis and wider use of SPECT imaging is recommended in dogs with thyroid neoplasia.

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.Significance:This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813

8658860258318000Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL, together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases, SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways.

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.

Health issues in purebred dogs are currently considered one of the biggest problems in companion animal health. The Labrador retriever (LR) is one of the most popular dog breeds. The aim of this study was to quantify LR breed health in comparison with mixed-breed dogs (MB), by using four different data sources: a veterinary practice management system (appr. 35,000 unique individuals LR + MB), data from two animal insurance companies (appr. 15,500 and 4500 individuals respectively), and a histopathological laboratory (appr. 4000 individuals). After extensive recoding of the data, health parameters utilised to quantify breed health were longevity, frequency of practice visits and insurance expense claims, and diagnostic codes. A Kaplan-Meier univariate and multivariable Cox proportional hazard model were used to evaluate longevity. A negative binomial model was used to analyse the frequency of visits, claims, and diagnostic codes in both sets of insurance data. Logistic regression was used to look into the categorical diagnostic codes in the laboratory data. The median lifespan of the LR was similar (12 years, practice data) or longer (10 versus 8 years, insurance data) than MB for individuals with a known birth and death date. When including censored individuals, survival time in the LR was comparable to MB individuals up to 10 years of age. Above 10 years of age, the LR lived a similar length as MB with a medium to large body size, but shorter than all MB. The LR visited the veterinary practice more often (risk ratio (RR) 1.2, 95% confidence interval 1.2-1.3), and also showed a higher frequency of insurance expense claims (RR 2.2 (2.1-2.3) and RR 1.2 (1.1-1.3) respectively for the two insurance data sets). The largest difference in organ systems between the LR and MB in insurance claims was related to ears (RR 5.3 (4.8-5.8) and RR 2.6 (2.3-3.1)), followed by airways (RR 2.6 (2.4-2.8)), tendons & muscles (RR 2.4 (2.2-2.6) and RR 1.4 (1.1-1.7)), and joints (RR 1.7 (1.3-2.1)), without a difference in median age at diagnosis. The data from the histopathological laboratory suggested a higher disease burden related to oncology for the LR compared to MB (OR 1.2, 95% CI 1.0-1.3). Oncological diagnoses were made at a younger age in the LR (8.8 versus 9.4 years). The disease burden was significantly higher for the LR than MB, but these results may suffer from substantial bias such as selection bias towards the database, and different behaviour of LR versus MB owners with regards to veterinary care. In the future, longer term population data can corroborate these results.

Reliable incidence measurement of diseases is necessary for identification of hereditary diseases in companion animal populations. The data collection system 'PETscan' was developed to facilitate standardized registration of diagnoses in veterinary practice. In the development, we attempted to counter challenges known from other primary practice data systems. PETscan includes a comprehensive list of potential diagnoses and supports the veterinary professionals in the diagnostic process. Demographics, individual data and standardized diagnostic data are collected through practice management software in a central database for epidemiological analysis. A preliminary data analysis from PETscan showed specific health issues in four canine breeds. As a real-time prospective monitoring tool, PETscan summaries can objectively assess the incidence of disorders in companion animal populations and can be used to prioritize disease-gene identification studies and evaluate the effects of breeding strategies, for example, after implementation of a new DNA test in the breeding strategy.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non-ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

Introduction To increase the efficacy of chemoradiation and decrease its toxicity in normal tissue, a new concept is proposed, local radiosensitizer delivery, which combines triggered release of a radiosensitizer from thermosensitive liposomes with local hyperthermia and radiotherapy. Here, key aspects of this concept were investigated in vitro I) the effect of hyperthermia on the enhancement of radiotherapy by ThermoDox (thermosensitive liposome containing doxorubicin), II) the concentration dependence of the radiosensitizing effect of doxorubicin and III) the sequence of doxorubicin, hyperthermia and radiotherapy maximizing the radiosensitizing effect. Methods Survival of HT1080 (human fibrosarcoma) cells was measured after exposure to ThermoDox or doxorubicin for 60 minutes, at 37 or 43°C, with or without irradiation. Furthermore, cell survival was measured for cells exposed to different doxorubicin concentrations and radiation doses. Finally, cell survival was measured after applying doxorubicin and/or hyperthermia before or after irradiation. Cell survival was measured by clonogenic assay. In addition, DNA damage was assessed by γH2AX staining. Results Exposure of cells to doxorubicin at 37°C resulted in cell death, but exposure to ThermoDox at 37°C did not. In contrast, ThermoDox and doxorubicin at 43°C resulted in similar cytotoxicity, and in combination with irradiation caused a similar enhancement of cell kill due to radiation. Doxorubicin enhanced the radiation effect in a small, but significant, concentration-dependent manner. Hyperthermia showed the strongest enhancement of radiation effect when applied after irradiation. In contrast, doxorubicin enhanced radiation effect only when applied before irradiation. Concurrent doxorubicin and hyperthermia immediately before or after irradiation showed equal enhancement of radiation effect. Conclusion In vitro, ThermoDox resulted in cytotoxicity and enhancement of irradiation effect only in combination with hyperthermia. Therefore hyperthermia-triggered radiosensitizer release from thermosensitive liposomes may ultimately serve to limit toxicities due to the radiosensitizer in unheated normal tissue and result in enhanced efficacy in the heated tumor.

Human epidermal growth factor 2 (HER2) overexpression leads to aggressive mammary tumour growth. Although the prognosis of HER2+ tumours in humans is greatly improved using biologicals, therapy resistance, which may be caused by increased phosphatidyl-3-kinase (PI3K), rous sarcoma proto-oncogene (cSRC) or wingless-type MMTV integration site family (Wnt) activity, is a major concern. A recent analysis of 12 canine mammary cell lines showed an association between HER2/3 overexpression and phosphatase and tensin homologue (PTEN) deletion with elevated Wnt-signalling. Wnt-activity appeared to be insensitive to phosphatidyl-3-kinase (PI3K) inhibitors but sensitive to Src-I1. We hypothesized that Wnt activation, was caused by HER2/3-activated cSRC activation. The role of HER2/3 on Wnt signalling was investigated by silencing HER2/3 expression using specific small interfering RNA (siRNAs). Next, the effect of an epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor on Wnt activity and migration was investigated and compared to other tyrosine kinase inhibitors (TKIs) of related signalling pathways. Finally, two TKIs, a cSRC and a PI3K inhibitor, were investigated in a zebrafish xenograft model. Silencing of HER1-3 did not inhibit the intrinsic high Wnt activity, whereas the HER kinase inhibitor afatinib showed enhanced Wnt activity. The strongest inhibition of Wnt activity and cell viability and migration was shown by cSRC inhibitors, which also showed strong inhibition of cell viability and metastasis in a zebrafish xenograft model. HER2/3 overexpression or HER2/3-induced cSRC activation is not the cause of enhanced Wnt activity. However, inhibition of cSRC resulted in a strong inhibition of Wnt activity and cell migration and metastasis. Further studies are needed to unravel the mechanism of cSRC activation and cSRC inhibition to restore sensitivity to HER-inhibitors in HER2/3-positive breast cancer.

A cortisol-secreting adrenocortical tumour (ACT) is the cause of naturally occurring canine hypercortisolism in approximately 15-20% of cases. The differentiation between an adrenocortical adenoma and carcinoma is usually based on histopathology. However, histopathological parameters have never been linked to the dogs' survival. Moreover, in human medicine the interobserver variability of some histopathological parameters that are used for ACTs is high. The objective of this study was to establish a reliable and easy-to-use histopathological scoring system for cortisol-secreting ACTs that can assess the prognosis of dogs after adrenalectomy. Cortisol-secreting ACTs of 50 dogs, collected between 2002 and 2015, were included in this study. Twenty histopathological features were assessed by one veterinary pathologist and one resident in veterinary pathology. In addition, the Ki67 proliferation index was assessed by two observers. Only parameters with intra- and interobserver agreement scores (intraclass correlation or Cohen's kappa coefficient) of ≥0.40 were included in survival analyses. Use of multivariate forward stepwise regression analysis with associated hazard ratios led us to a scoring system which we call the Utrecht score: the Ki67 proliferation index, +4 if more than 33% of the tumour cells have clear/vacuolated cytoplasm, and +3 if necrosis is present. Using cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing Utrecht scores. We conclude that the Utrecht score can be used to assess the prognosis of dogs with cortisol-secreting ACTs after adrenalectomy, which can help to select high-risk dogs that might benefit from adjuvant treatment or additional monitoring. This article is protected by copyright. All rights reserved.

Objective: Inhibition of GH signaling has been associated with increased longevity and cancer resistance. As the fetal estrogen estetrol was also linked to longevity and cancer resistance, we hypothesized that these effects of estetrol are mediated by inhibition of GH activity. Design: The CNMm-6TR mammary cell line, stably transfected with doxycycline (DOX)-inducible GHR (CNMm-cGHR cells), HEK293 cell line transfected with rGHR and the human T47D cell line were used to assess effects of estetrol on GH signal transduction. Proliferation was measured using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT). Spi-luciferase reporter activity, responsive to phosphorylated Stat5, was used to assess GH signaling. GHR mRNA expression was normalized to a reference gene. Results: Treatment of CNMm-cGHR cells with GH resulted in decreased proliferation rates. No effect was seen by the co-inhibition with estetrol. Using the spi-luc reporter activity, it was shown that GH activates the GHR/Jak2/ Stat5 pathway. Estetrol partially inhibited this activation. In the HEK-rGHR cells, estetrol was able to inhibit the GH induced activation of the spi-luc reporter, but only in the presence of an ERα expression plasmid. Effects of estetrol on the expression of GHR mRNA was assessed in T47D cells, a cell-line expressing ER, PR and GHR under natural promoters. After incubation with estetrol, a mean 4-fold inhibition of GHR mRNA expression was observed. Conclusion: Using different cell systems, it was shown that estetrol is able to inhibit GH signaling, a process dependent on the presence of the Estrogen Receptor (ER). Estetrol may therefore be used to inhibit GH-induced effects on breast cancer proliferation.

Mammary tumors are the most common form of neoplasia in the bitch. Female dogs are protected when they are spayed before the first estrus cycle, but this effect readily disappears and is already absent when dogs are spayed after the second heat. As the ovaries are removed during spaying, ovarian steroids are assumed to play an essential role in tumor development. The sensitivity toward tumor development is already present during early life, which may be caused by early mutations in stem cells during the first estrus cycles. Later on in life, tumors arise that are mostly steroid-receptor positive, although a small subset of tumors overexpressing human epidermal growth factor 2 (HER2) and some lacking estrogen receptor, progesterone receptor (PR), and HER2 (triple negative) are present, as is the situation in humans. Progesterone (P4), acting through PR, is the major steroid involved in outgrowth of mammary tissue. PRs are expressed in two forms, the progesterone receptor A (PRA) and progesterone receptor B (PRB) isoforms derived from splice variants from a single gene. The dog and the whole family of canids have only a functional PRA isoform, whereas the PRB isoform, if expressed at all, is devoid of intrinsic biological activity. In human breast cancer, overexpression of the PRA isoform is related to more aggressive carcinomas making the dog a unique model to study PRA-related mammary cancer. Administration of P4 to adult dogs results in local mammary expression of growth hormone (GH) and wing less-type mouse mammary tumor virus integration site family 4 (Wnt4). Both proteins play a role in activation of mammary stem cells. In this review, we summarize what is known on P4, GH, and Wnt signaling in canine mammary cancer, how the family of HER receptors could interact with this signaling, and what this means for comparative and translational oncological aspects of human breast cancer development.

Background In the last decade, a disorder characterized by episodes of involuntary movements and dystonia has been recognized in Border Terriers. Objectives To define clinical features of paroxysmal dyskinesia (PD) in a large number of Border Terriers and to study the genetics of the disease. Animals 110 affected and 128 unaffected client-owned Border Terriers. Methods A questionnaire regarding clinical characteristics of PD was designed at Utrecht University and the University of Helsinki. Thirty-five affected Border Terriers underwent physical examination and blood testing (hematology and clinical biochemistry). Diagnostic imaging of the brain was performed in 17 affected dogs and electroencephalograms (EEG) between episodes were obtained in 10 affected dogs. A genomewide association study (GWAS) was performed with DNA of 110 affected and 128 unaffected dogs. Results One hundred forty-seven questionnaires were included in the study. The most characteristic signs during episodes were dystonia, muscle fasciculations, and falling over. The majority of owners believed that their dogs remained conscious during the episodes. A beneficial effect of anti-epileptic therapy was observed in 29 of 43 dogs. Fifteen owners changed their dogs’ diet to a hypoallergenic, gluten-free diet, and all reported reasonable to good improvement of signs. Clinical examinations and diagnostic test results were unremarkable. The GWAS did not identify significantly associated chromosome regions. Conclusions and Clinical Importance The survey results and EEG studies provided further evidence that the observed syndrome is a PD rather than epilepsy. Failure to achieve conclusive results by GWAS indicates that inheritance of PD in Border Terriers probably is complex.

Background Transsphenoidal hypophysectomy is one of the treatment strategies in the comprehensive management of dogs with pituitary‐dependent hypercortisolism (PDH). Objectives To describe the influence of pituitary size at time of pituitary gland surgery on long‐term outcome. Animals Three‐hundred–and‐six dogs with PDH. Methods Survival and disease‐free fractions were analyzed and related to pituitary size; dogs with and without recurrence were compared. Results Four weeks after surgery, 91% of dogs were alive and remission was confirmed in 92% of these dogs. The median survival time was 781 days, median disease‐free interval was 951 days. Over time, 27% of dogs developed recurrence of hypercortisolism after a median period of 555 days. Dogs with recurrence had significantly higher pituitary height/brain area (P/B) ratio and pre‐operative basal urinary corticoid‐to‐creatinine ratio (UCCR) than dogs without recurrence. Survival time and disease‐free interval of dogs with enlarged pituitary glands was significantly shorter than that of dogs with a non‐enlarged pituitary gland. Pituitary size at the time of surgery significantly increased over the 20‐year period. Although larger tumors have a less favorable prognosis, outcome in larger tumors improved over time. Conclusions and Clinical Importance Transsphenoidal hypophysectomy is an effective treatment for PDH in dogs, with an acceptable long‐term outcome. Survival time and disease‐free fractions are correlated negatively with pituitary gland size, making the P/B ratio an important pre‐operative prognosticator. However, with increasing experience, and for large tumors, pituitary gland surgery remains an option to control the pituitary mass and hypercortisolism.

BACKGROUND & AIMS: A "microbrachytherapy" was developed as treatment option for inoperable tumours by direct intratumoral injection of radioactive holmium-166 ( (166) Ho) microspheres (MS). (166) Ho emits β-radiation which potentially enables a high, ablative, radioactive-absorbed dose on the tumour tissue while sparing surrounding tissues. MATERIALS & METHODS: Safety and efficacy of (166) Ho microbrachytherapy were evaluated in a prospective cohort study of 13 cats with inoperable oral squamous cell carcinoma without evidence of distant metastasis. RESULTS: Local response rate was 55%, including complete response or partial response (downstaging) enabling subsequent marginal resection. Median survival time was 113 days overall, and 296 days for patients with local response. Side effects were minimal. Tumour volume was a significant predictor of response. DISCUSSION: Response rate may be further improved by optimizing the intratumoral spatial distribution of (166) Ho MS. CONCLUSION: (166) Ho microbrachytherapy has potential as a minimally invasive, single procedure radio-ablation treatment of unresectable tumours with minimal morbidity.

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASCs application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using adipose tissue-derived cell (ASC) sheet transplantation therapy for promoting tissue healing. Firstly, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion, and capillary density and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the gastrointestinal system accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these pre-clinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental set-ups according to the SYRCLE's guidelines should improve study quality and validity.

Canine hypercortisolism is most often caused by an ACTH-secreting pituitary adenoma (pituitary-dependent hypercortisolism; PDH). An interesting target for a selective medical treatment of PDH would be the receptor for ACTH: the melanocortin 2 receptor (MC2R). In this study we investigated whether two peptide compounds, BIM-22776 (#776) and BIM-22A299 (#299), are effective MC2R antagonists in vitro. Their effects on cortisol production and mRNA expression of steroidogenic enzymes, MC2R and melanocortin 2 receptor accessory protein (MRAP) were evaluated in primary adrenocortical cell cultures (n = 8) of normal canine adrenal glands. Cortisol production stimulated by 50 nM ACTH was dose-dependently inhibited by #299 (inhibition 90.7 ± 2.3% at 5 μM) and by #776 (inhibition 38.0 ± 5.2% at 5 μM). The ACTH-stimulated mRNA expression of steroidogenic enzymes, MC2R and MRAP was significantly inhibited by both compounds, but most potently by #299. These results indicate that canine primary cell culture is a valuable in vitro system to test MC2R antagonists, and that these compounds, but especially #299, are effective MC2R antagonists in vitro. To determine its efficacy in vivo, further studies are warranted. Antagonism of the MC2R is a promising potential treatment approach in canine PDH.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are described in several species. In humans and dogs they have many similarities: the excessive catecholamine release in hormonally active PCC causes similar clinical signs, the frequency of metastasis is similar, and they are histopathologically almost identical. Surgery is curative when PCC and PGL have not metastasized, while only palliative treatment is possible for patients with metastatic disease. Mutations in succinate dehydrogenase subunit B (SDHB) are associated with metastatic behaviour in human PCC/PGL and the same mutation has been described in dogs. The dog might therefore be a suitable model for study of the pathogenesis of metastatic PCC and PGL in humans. Further molecular studies of common tumourigenic pathways and comparative studies of histopathology of human and canine PCC and PGL are warranted.

Background Quantitative PCR (qPCR) is a common method for quantifying mRNA expression. Given the heterogeneity present in tumor tissues, it is crucial to normalize target mRNA expression data using appropriate reference genes that are stably expressed under a variety of pathological and experimental conditions. No studies have validated specific reference genes in canine osteosarcoma (OS). Previous gene expression studies involving canine OS have used one or two reference genes to normalize gene expression. This study aimed to validate a panel of reference genes commonly used for normalization of canine OS gene expression data using the geNorm algorithm. qPCR analysis of nine canine reference genes was performed on 40 snap-frozen primary OS tumors and seven cell lines. Results Tumors with a variety of clinical and pathological characteristics were selected. Gene expression stability and the optimal number of reference genes for gene expression normalization were calculated. RPS5 and HNRNPH were highly stable among OS cell lines, while RPS5 and RPS19 were the best combination for primary tumors. Pairwise variation analysis recommended four and two reference genes for optimal normalization of the expression data of canine OS tumors and cell lines, respectively. Conclusions Appropriate combinations of reference genes are recommended to normalize mRNA levels in canine OS tumors and cell lines to facilitate standardized and reliable quantification of target gene expression, which is essential for investigating key genes involved in canine OS metastasis and for comparative biomarker discovery.

The long-term prognosis after surgical resection of malignant insulinoma (INS) is poor. Novel adjuvant therapies, specifically targeting cancer stem cells (CSCs), are warranted. Therefore, the goal of this study was to characterize and target putative INS CSCs. Using fluorescence-activated cell sorting, human INS cell line CM and pancreatic carcinoid cell line BON1 were screened for the presence of stem cell-associated markers. CD90, CD166, and GD2 were identified as potential CSC markers. Only CD90(+) INS cells had an increased tumor-initiating potential in athymic nude mice. Anti-CD90 monoclonal antibodies decreased the viability and metastatic potential of injected cells in a zebrafish embryo INS xenograft model. Primary INS stained positive for CD90 by immunohistochemistry, however also intratumoral fibroblasts and vascular endothelium showed positive staining. The results of this study suggest that anti-CD90 monoclonals form a potential novel adjuvant therapeutic modality by targeting either INS cells directly, or by targeting the INS microenvironment.

Morphologically, canine soft-tissue sarcomas (STSs) resemble human STSs. In humans, proper classification of STSs is considered essential to improve insight in the biology of these tumors, and to optimize diagnosis and therapy. To date, there is a paucity of data published on the significance of detailed classification of STSs in the dog. We revised a cohort ( n = 110) of proliferative lesions obtained from a study in Golden Retrievers that were considered “soft tissue sarcoma, not otherwise specified or of uncertain subtype” in order to optimize the diagnoses of these lesions. The criteria according to the veterinary WHO classification, recent veterinary literature, and the WHO classification for humans were applied. Revision was initially based on morphologic characteristics of hematoxylin and eosin–stained histologic sections of the neoplasms. If considered necessary ( n = 76), additional immunohistochemistry was applied to aid characterization. The diagnosis of STS was confirmed in 75 neoplasms (68%). Of this group, diagnosis of a specific subtype of the STSs was possible in 58 neoplasms. Seven neoplasms had morphologic characteristics that were suggestive for sarcoma subtypes only described in the WHO classification for humans. Seventeen neoplasms remained “unclassified STSs.” Thirty-one lesions (28%) were diagnosed “neoplasm, not being STS.” Four lesions (4%) were considered nonneoplastic. Because incorrect classification of a tumor could lead to inappropriate therapeutic intervention and prognostication, the results of our study clearly illustrate the importance of revision and further diagnosis of “unclassified STSs” in dogs.

International audience; BackgroundSerum cystatin C (sCysC) is used as biomarker for glomerular filtration rate (GFR). The effects of diabetes mellitus (DM) on renal function in dogs are unclear. Some renal variables have been evaluated in dogs with hyperadrenocorticism (HAC), but not sCysC. ObjectivesThe purpose of this study was the validation of a particle-enhanced nephelometric immunoassay (PENIA) for measuring canine sCysC, and to assess renal function in dogs with DM or HAC. MethodsA PENIA was analytically validated for canine sCysC by determining imprecision and linearity. In a longitudinal 6-month study, renal function of 14 DM dogs was assessed, using serum creatinine, GFR, urinary protein-to-creatinine (UPC) ratio, urinary markers, systolic blood pressure (SBP), and sCysC, and compared to 17 healthy dogs at baseline. Furthermore, sCysC was measured at initial presentation and during a 12-month follow-up in 22 HAC dogs. ResultsThe sCysC intra- and inter-assay variation coefficients were < 8% and highly linear (r = .997). About 33% and 67% of DM dogs had persistent proteinuria and systemic hypertension, respectively, but there were no significant differences in GFR, UPC, and urinary markers over time, and compared with healthy dogs at initial presentation. Serum CysC decreased significantly (P < .05) over time within the DM group. It did not change significantly over time within the HAC group. ConclusionsA PENIA measured sCysC linearly and precisely. There were no clinically relevant renal alterations over time in dogs with DM, although persistent proteinuria was observed. In dogs with HAC, sCysC measurement was not useful, although significant GFR changes occurred over time.

Spontaneous cortisol-secreting adrenocortical tumours in pet dogs are an attractive animal model for their human counterparts. Adrenal morphology and function are similar in dogs and humans, and adrenocortical tumours have comparable clinical and pathological characteristics. Their relatively high incidence in pet dogs represents a potential source of adrenocortical tumour tissue to facilitate research. The molecular characteristics of canine cortisol-secreting adrenocortical tumours suggest that they will be useful for the study of angiogenesis, the cAMP/protein kinase A pathway, and the role of Steroidogenic Factor-1 in adrenal tumourigenesis. Pet dogs with spontaneous cortisol-secreting adrenocortical tumours may also be useful in clinical testing of new drugs and in investigating the molecular background of adrenocortical tumours.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo. Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267–81. ©2017 AACR.

Adipose tissue-derived stem cells (ASCs) are known to be able to promote repair of injured tissue via paracrine factors. However, the effect of cell density and inflammatory cytokines on the paracrine ability of ASCs remains largely unknown. To investigate these effects, ASCs were cultured in 8000 cells/cm2, 20,000 cells/cm2, 50,000 cells/cm2, and 400,000 cells/cm2 with and without 10 or 20 ng/ml tumor necrosis factor alpha (TNFα) and 25 or 50 ng/ml interferon gamma (IFNγ). ASC-sheets formed at 400,000 cells/cm2 after 48 h of culture. With increasing concentrations of TNFα and IFNγ, ASC-sheets with 400,000 cells/cm2 had increased production of angiogenic factors Vascular Endothelial Growth Factor and Fibroblast Growth Factor and decreased expression of pro-inflammatory genes TNFA and Prostaglandin Synthase 2 (PTGS2) compared to lower density ASCs. Moreover, the conditioned medium of ASC-sheets with 400,000 cells/cm2 stimulated with the low concentration of TNFα and IFNγ enhanced endothelial cell proliferation and fibroblast migration. These results suggest that a high cell density enhances ASC paracrine function might beneficial for wound repair, especially in pro-inflammatory conditions. Electronic supplementary material The online version of this article (doi:10.1007/s12015-017-9719-3) contains supplementary material, which is available to authorized users.

RATIONALE: Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. OBJECTIVES: To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. METHODS: Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. RESULTS: The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. CONCLUSIONS: Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.

This study evaluated efficacy and side effects of masitinib in canine epitheliotropic lymphoma. Complete remission occurred in 2 of 10 dogs and lasted for median 85 days. Five dogs went into partial remission for median 60.5 days. Three pretreated dogs did not respond to therapy. Side effects occurred in six dogs and were mostly mild to moderate. Immunohistochemistry was available for eight dogs. KIT receptor was negative in all of them, six of eight lymphomas stained strongly positive for stem cell factor (SCF). platelet-derived growth factor (PDGF)-AA was weakly positive in two and negative in six. PDGF-BB was negative in four tumours, weakly positive in one and strongly positive in three. One was strongly positive for PDGF receptor (PDGFR)-β, seven were negative for that receptor. Five showed strong expression of PDGFR-α, two showed weak expression, one was negative. In conclusion, masitinib is effective in treating canine epitheliotropic lymphoma. But its effects are most likely not generated through the KIT receptor.

Cortisol-secreting adrenocortical tumours (AT) in dogs are characterised by uncontrolled growth and excessive cortisol secretion. Dysregulated hormone receptor expression might be involved in tumour growth and hypersecretion of cortisol. The relative mRNA expression of growth hormone receptor, progesterone receptor, somatostatin receptors (SSTR1-3) and dopamine receptors (DRD1-2 and DRD5) was evaluated in 36 canine ATs and 15 adrenal glands obtained from healthy dogs. Compared with normal adrenal tissue, DRD2 mRNA expression was relatively lower in carcinomas, while SSTR1 mRNA expression was lower in both adenomas and carcinomas. Both of these features might contribute to loss of inhibition of tumour growth and upregulation of cortisol secretion. In canine ATs that had recurred within 30 months of surgical adrenalectomy, a marked increase in expression of DRD1 mRNA was observed. Targeting of specific hormone receptors, expressed by ATs, might be exploited for therapy.

Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and tumor-selective treatment, using nanobodies (NBs) to deliver a photosensitizer (PS) specifically to cancer cells. Upon local light application, reactive oxygen species are formed and consequent cell death occurs. NB-PDT has preclinically shown evident success and we next aim to treat cats with oral squamous cell carcinoma (OSCC), which has very limited therapeutic options and is regarded as a natural model of human head and neck SCC. Immunohistochemistry of feline OSCC tissue confirmed that the epidermal growth factor receptor (EGFR) is a relevant target with expression in cancer cells and not in the surrounding stroma. Three feline OSCC cell lines were employed together with a well-characterized human cancer cell line (HeLa), all with similar EGFR expression, and a low EGFR-expressing human cell line (MCF7), mirroring the EGFR expression level in the surrounding mucosal stroma. NBA was identified as a NB binding human and feline EGFR with comparable high affinity. This NB was developed into NiBh, a NB-PS conjugate with high PS payload able to effectively kill feline OSCC and HeLa cell lines, after illumination. Importantly, the specificity of NB-PDT was confirmed in co-cultures where only the feline OSCC cells were killed while surrounding MCF7 cells were unaffected. Altogether, NiBh can be used for NB-PDT to treat feline OSCC and further advance NB-PDT towards the human clinic.

Hypercortisolism is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15-20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol-secreting ACTs were included from 40 dogs, of which follow-up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate-high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT-PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour-transforming gene-1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor-1 (SF-1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets. This article is protected by copyright. All rights reserved.

Pseudopregnancy is a frequently diagnosed reproductive disorder in (dairy) goats. This cross-sectional study evaluates the incidence, possible risk factors and therapies for pseudopregnancy on Dutch dairy goat farms. Two questionnaires, one for farmers and one for veterinarians, were designed and included questions about general farm demographics, breeding management, hormonal oestrous induction, treatment, measures for reduction and stress moments in dairy goats in the period June 1, 2016-May 31, 2017. In total, 43 farmers (21.5 per cent response rate) and 27 veterinarians (22.5 per cent response rate) completed the questionnaire. The annual incidence of pseudopregnancy varied between 1 and 54 per cent per farm, with a mean annual incidence of 17 per cent (95 per cent CI 0.14 to 0.21). In this study, we found a significant association between incidence of pseudopregnancy and a higher percentage of goats with an extended lactation (p

Naturally occurring hypercortisolism, also known as Cushing's syndrome, is a common endocrine disorder in dogs that can be caused by an adenocorticotrophic hormone (ACTH)-producing pituitary adenoma (pituitary-dependent hypercortisolism, PDH; 80–85% of cases), or by an adrenocortical tumor (ACT; 15–20% of cases). To determine the optimal treatment strategy, differentiating between these two main causes is essential. Good treatment options are surgical removal of the causal tumor, i.e. hypophysectomy for PDH and adrenalectomy for an ACT, or radiotherapy in cases with PDH. Because these options are not without risks, not widely available and not suitable for every patient, pharmacotherapy is often used. In cases with PDH, the steroidogenesis inhibitor trilostane is most often used. In cases with an ACT, either trilostane or the adrenocorticolytic drug mitotane can be used. Although mostly effective, both treatments have disadvantages. This review discusses the current treatment options for canine hypercortisolism, and considers their mechanism of action, efficacy, adverse effects, and effect on survival. In addition, developments in both adrenal-targeting and pituitary-targeting drugs that have the potential to become future treatment options are discussed, as a more selective and preferably also tumor-targeted approach could have many advantages for both PDH and ACTs.

Objective: Inhibition of GH signaling has been associated with increased longevity and cancer resistance. As the fetal estrogen estetrol was also linked to longevity and cancer resistance, we hypothesized that these effects of estetrol are mediated by inhibition of GH activity. Design: The CNMm-6TR mammary cell line, stably transfected with doxycycline (DOX)-inducible GHR (CNMm-cGHR cells), HEK293 cell line transfected with rGHR and the human T47D cell line were used to assess effects of estetrol on GH signal transduction. Proliferation was measured using the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT). Spi-luciferase reporter activity, responsive to phosphorylated Stat5, was used to assess GH signaling. GHR mRNA expression was normalized to a reference gene. Results: Treatment of CNMm-cGHR cells with GH resulted in decreased proliferation rates. No effect was seen by the co-inhibition with estetrol. Using the spi-luc reporter activity, it was shown that GH activates the GHR/Jak2/ Stat5 pathway. Estetrol partially inhibited this activation. In the HEK-rGHR cells, estetrol was able to inhibit the GH induced activation of the spi-luc reporter, but only in the presence of an ERα expression plasmid. Effects of estetrol on the expression of GHR mRNA was assessed in T47D cells, a cell-line expressing ER, PR and GHR under natural promoters. After incubation with estetrol, a mean 4-fold inhibition of GHR mRNA expression was observed. Conclusion: Using different cell systems, it was shown that estetrol is able to inhibit GH signaling, a process dependent on the presence of the Estrogen Receptor (ER). Estetrol may therefore be used to inhibit GH-induced effects on breast cancer proliferation.

RATIONALE: Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. OBJECTIVES: To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. METHODS: Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. RESULTS: The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. CONCLUSIONS: Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASCs application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using adipose tissue-derived cell (ASC) sheet transplantation therapy for promoting tissue healing. Firstly, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion, and capillary density and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the gastrointestinal system accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these pre-clinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental set-ups according to the SYRCLE's guidelines should improve study quality and validity.

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer, but could also be an interesting treatment of Cushing's syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM - 10 μM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In conclusion, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model.

Epithelial cancers are defined by a tumor-specific distribution of chromosomal aneuploidies that are maintained when cells metastasize and are conserved in cell lines derived from primary tumors. Correlations between genomic copy number and gene expression have been observed for different tumors including, colorectal (CRC), breast and pancreatic cancer. These ploidy-driven transcriptional deregulations are characterized by low-level expression changes of most genes on the affected chromosomes. The emergence of these aberrations at an early stage of tumorigenesis and the strong selection for the maintenance of these aneuploidies suggests that aneuploidy-dependent transcriptional deregulations might contribute to cellular transformation and maintenance of the malignant phenotype. The histone deacetylase inhibitor (HDACi) Trichostatin A (TSA) has anticancer effects and is well known to lead to large scale gene expression changes. Here we assessed if TSA could disrupt the aneuploidy-driven gene expression in the aneuploid colon cancer cell line SW480 and the artificially generated aneuploid cell line DLD-1 + 7. We found that TSA increases transcriptional activity throughout the genome, yet inhibits aneuploidy-induced gene expression changes on chromosome 7. Among the TSA affected genes on chromosome 7 we identified potential CRC oncogenes. These experiments represent the first attempt to explain how histone acetylation affects aneuploidy-driven gene expression changes. This article is protected by copyright. All rights reserved.