Takuya Maruo

Identification of Cancer Stem Cells Derived From a Canine Lung Adenocarcinoma Cell Line

Accumulating evidence supporting the cancer stem cell (CSC) hypothesis is based on the finding that tumors contain a small population of self-renewing cells that generate differentiated progeny and thereby contribute to tumor heterogeneity. CSCs are reported to exist in several human cancers, yet only a few reports demonstrate the existence of CSCs in primary lung cancer in dogs. In this study, the authors established a cancer cell line derived from a canine primary lung adenocarcinoma and identified a side population (SP) of cells that displayed drug-resistant features. To confirm the characteristics of these SP cells, the authors investigated the tumorigenicity of the cells in vivo by using a nude mouse xenograft model. Only 100 SP cells were able to give rise to new tumors, giving a 10-fold enrichment over the main population (MP) of cells, suggesting that these cells have the cancer-initiating ability of CSCs. Further studies characterizing CSCs in canine lung adenocarcinoma might contribute to the elucidation of the mechanisms of tumorigenesis and to the establishment of novel therapeutic strategies.

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.

The palliative efficacy of modified Mohs paste for controlling canine and feline malignant skin wounds

ABSTRACT In veterinary medicine, the management of malignant skin wounds is highly challenging. We conducted a study on seven case animals (four dogs and three cats) which presented with malignant skin wounds. All seven animals had signs and symptoms which were controlled following treatment with a modified Mohs paste. Upon obtaining informed consent from their owners, the animals requiring management of malignant wounds were enrolled in this study. The modified Mohs paste was prepared by mixing zinc chloride, zinc oxide starch powder, glycerin, and distilled water. The modified Mohs paste was topically applied to and left to remain on the malignant wounds for one hour, under controlled conditions. Once the paste was removed, the wounds were irrigated with a solution of sterile saline. At the first examination, the wounds of each animal were observed for signs of exudate, malodor, and bleeding. In every case, visible improvement was observed immediately after the modified Mohs paste treatment. Specifically, the size of the malignant wounds, and the number of times the dressing gauze required changing, significantly decreased (p < 0.05 and p < 0.01, respectively). The open malignant skin wounds caused by mammary gland tumors disappeared in two cases. The Mohs paste has been shown to be a viable option for the palliative treatment in canine and feline malignant skin wound management.

VALIDATION OF NEW BITE BLOCK-TYPE HEAD-IMMOBILIZATION DEVICES FOR RADIOTHERAPY IN DOGS

An ideal head-immobilization method provides a high level of accuracy and reproducibility in the immobilization. Various head-immobilization methods for radiotherapy have been published and are excellent in terms of accuracy; however, these methods are complicated to use, and labor intensive. The present study describes two new bite block-type head-immobilization devices designed for higher stability and lower vertical variation. The device designed in our previous study (the bite block-type head-immobilization device; Device A) was modified by making a groove on the top the horizontal plate (Device B) for a stable ventral-dorsal position, or beneath the horizontal plate (Device C) for a stable dorsal-ventral position. The three devices were objectively compared with respect to setup time, and accuracy of the computed tomography scan images by two authors independently. Five male healthy beagles were used in this study. For each device, the setup time and the variation in the coordinates were measured five times for each dog. The mean setup times for Devices A, B, and C were 3.3, 1.5, and 2.4 min, respectively, showing the groove modifications were able to reduce the setup time (in device B, by at least 50%). Moreover, three-dimensional analysis of the computed tomography images revealed that the measurement variability of Device A (1.6 ± 1.0 mm) was significantly higher than that of Device C (0.7 ± 0.4 mm; P < 0.001). Collectively, our results show that use of a bite block-type head-immobilization device with a groove improves the setup time and head-immobilization accuracy.

Booster effect of canine distemper, canine parvovirus infection and infectious canine hepatitis combination vaccine in domesticated adult dogs

Domesticated adult dogs with antibody titer classified as below ‘high’ to one or more of canine distemper virus (CDV), canine parvovirus type‐2 (CPV‐2) and canine adenovirus type‐1 (CAdV‐1) were then given an additional inoculation, and the effectiveness of this booster evaluated 2 months later. Consequently, CDV and CAdV‐1 antibody titer experienced a significant increase, but the same effect was not observed in the antibody titer of CPV‐2. These findings suggest that with additional inoculation, a booster effect may be expected in increasing antibody titers for CDV and CAdV‐1, but it is unlikely to give an increase in CPV‐2 antibody titer.

Canine Amino Acid Transport System Xc–: cDNA Sequence,Distribution and Cystine Transport Activity in Lens Epithelial Cells

ABSTRACT The cystine transport activity of a lens epithelial cell line originated from a canine mature cataract was investigated. The distinct cystine transport activity was observed, which was inhibited to 28% by extracellular 1 mM glutamate. The cDNA sequences of canine cysteine/glutamate exchanger (xCT) and 4F2hc were determined. The predicted amino acid sequences were 527 and 533 amino acid polypeptides, respectively. The amino acid sequences of canine xCT and 4F2hc showed high similarities (>80%) to those of humans. The expression of xCT in lens epithelial cell line was confirmed by western blot analysis. RT-PCR analysis revealed high level expression only in the brain, and it was below the detectable level in other tissues.

Antibodies to parvovirus, distemper virus and adenovirus conferred to household dogs using commercial combination vaccines containing Leptospira bacterin.

To examine how the inclusion (+) or exclusion (–) of inactivated Leptospira antigens in a vaccine for canine parvovirus type 2 (CPV-2), canine distemper virus (CDV) and canine adenovirus type 2 (CAdV-2) affects antibody titres to CPV-2, CDV and CAdV-1 antigens, household dogs were vaccinated with commercially available vaccines from one of three manufacturers. CPV-2, CDV and CAdV-1 antibody titres were measured 11 to 13 months later and compared within three different age groups and three different bodyweight groups. There were significant differences between CPV-2 antibody titres in dogs vaccinated with (+) vaccine and those vaccinated with (–) vaccine for two products in the two-year-old group and for one product in the greater than seven-year-old group; no significant differences were seen that could be attributed to bodyweight. No differences in CDV antibody titres were observed within age groups, but a significant difference was seen in the 11 to 20 kg weight group for one product. Significant differences in CAdV-1 antibody titres were seen for one product in both the two-year-old group and the ≤10 kg weight group.

A NOVEL SUPPORT DEVICE FOR HEAD IMMOBILIZATION DURING RADIATION THERAPY THAT IS APPLICABLE TO BOTH CATS AND DOGS.

Repeatable head immobilization is important for minimizing positioning error during radiation therapy for veterinary patients with head neoplasms. The purpose of this retrospective cross-sectional study was to describe a novel technique for head immobilization (Device II) and compare this technique with a previously described technique (Device I). Device II provided additional support by incorporating three teeth (vs. two teeth with Device I). Between 2011 and 2013, both devices were applied in clinically affected cats (Device I, n = 17; Device II, n = 11) and dogs (Device I, n = 85; Device II, n = 22) of various breeds and sizes. The following data were recorded for each included patient: variability in the angle of the skull (roll, yaw, and pitch), coordinates of the isocenter, and distance from the reference mark to the tumor. Devices I and II differed for skull angle variability during the treatment of dogs (roll, P = 0.0007; yaw, P = 0.0018; pitch, P = 0.0384) and for yaw of during the treatment of cats (P < 0.0001). In each case, Device II was superior to Device I. The distance from the reference mark to the center of the tumor was significantly decreased for Device II vs. Device I (dogs, P < 0.0001; cats, P = 0.0002). Device II also provided more accurate coordinates for the isocenter. Authors recommend the use of, Device II for future clinical patients.

Primary Lung Carcinoma with Paraneoplastic Leukocytosis in a Dog

In human lung cancers, marked leukocytosis, especially with neutrophilia and monocytosis, has been reported as a paraneoplastic syndrome. In dogs, several reports have been published on paraneoplastic leukocytosis. In this case, various examinations were conducted on a dog with extreme neutrophilic leukocytosis diagnosed via a blood test at first admission. A huge pulmonary mass was detected, which was surgically removed. The tumor was diagnosed as papillary adenocarcinoma of the lung by histopathology. The number of neutrophils recovered to normal after surgery, and the dog was diagnosed as paraneoplastic syndrome due to lung cancer. Key word:dog, leukocytosis, lung cancer, paraneoplastic syndrome 1)Sato Animal Hospital, 1-14 Miyashitachou, Ichinoseki, Iwate 021-0013, Japan 2)School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan 3)Veterinary Teaching Hospital, Azabu University, 1-17-71 Fuchinobe, Chuo, Sagamihara, Kanagawa 252-5201, Japan 4)Yamada Animal Hospital, 2-1, Koorikitanomachi, Neyagawa, Osaka 572-0080, Japan Corresponding author:Takuo Shida Telephone:042-754-7111 Facsimile:042-769-2418 Email address:shida@azabu-u.ac.jp Received:May 27, 2012 / Accepted:September 12, 2012 Note J Jpn Vet Cancer Soc, 3(2) −21− were all within normal ranges. According to these results, we surmised that the elevated BUN level was prerenal secondary to cardiac insufficiency. Based on these clinical data, the cause of the cough and the neutrophilia were considered to possibly be related to the thoracic mass. In order to improve the cough, thoracotomy was performed. The patient underwent surgery through the intercostal approach on the 15th day after first admission. The 5th rib of the patient was removed due to the large size of the mass. The mass involved the left caudal lung lobe and adhered to the pleura of the mediastinum and diaphragm. The pericardium was not involved in the mass. Blunt dissection of the mass from the pleura of the mediastinum caused some bleeding. After blunt dissection from the adhered sites, the left lung lobe was totally removed with the mass. Swelling of the tracheobronchial lymph nodes was not observed. The patient recovered from surgery without any events and showed good healthy conditions. The WBC count decreased to the normal range 2 weeks after surgery(Table 3)and was normal for 21 weeks after surgery. The patient’s cough disappeared after surgery, and the respiratory function of the patient was recovered. Furthermore, cardiac function was maintained well with the simultaneous administration of digoxin and an angiotensin converting enzyme inhibitor for cardiac insufficiency. However, cardiac function worsened, and 4 months after surgery, the patient had an increased BUN level. The patient showed collapse/syncope due to cardiac failure and died 5 months after surgery. Autopsy was not permitted by the client. Histopathological examination of the pulmonary mass revealed that the tissue consisted of multiple neoplastic foci divided by various amounts of fibrous septa. The neoplastic foci were characterized by papillary or glandular structures that were lined by cuboid to cylindroid epithelial cells. Some tumor cells showed a goblet-cell appearance, while some had cilia on their cell surface. These proliferating neoplastic cells showed moderate cellular atypia, and the mitotic figure index was 2-3 per high-power magnification(×400)field. The tumor cells invaded the lymph vessels of the surrounding tissues. Multifocal necrotic areas were also observed within the neoplastic foci. In the interstitial areas, there were moderate infiltrations of lymphocytes and macrophages, deposition of calcium, and occasional ossification. The pleurae were Fig. 1.Thoracic radiography(ventrodorsal view)revealed a large mass in the left caudal lung area that compressed and shifted the enlarged heart to the right. Fig. 2.Numerous neutrophils were observed in the blood smear specimen (×100).