Involvement of Nestin in the Progression of Canine Mammary Carcinoma.

Nestin, a class VI intermediate filament protein, is known to be expressed in various types of human neoplasms, including breast cancer, and is associated with their progression. However, its expression and role in canine mammary tumors remain unknown. We analyzed nestin expression in canine mammary tumors using in situ hybridization and immunohistochemistry. We also investigated its role in a canine mammary carcinoma cell line using RNA interference. Nestin expression was not observed in luminal epithelial cells of any of the 62 cases of benign mammary lesions examined, although myoepithelial cells showed its expression in most cases. In 16/50 (32%) primary mammary carcinomas and 6/15 (40%) metastases of mammary carcinomas, cytoplasmic nestin expression was detected in luminal epithelial cells. In luminal cells of primary mammary carcinomas, its expression was positively related to several pathological parameters that indicate high-grade malignancy, including histological grading (P < .01), vascular/lymphatic invasion (P < .01), Ki-67 index (P < .01), and metastasis (P < .05). Immunohistochemistry revealed that nestin expression was related to vimentin expression in mammary carcinomas (P < .01). This relationship was confirmed using reverse transcription-quantitative polymerase chain reaction using 9 cell lines derived from canine mammary carcinoma (P < .01). Finally, nestin knockdown in canine mammary carcinoma cells using small interfering RNA inhibited cell proliferation and migration based on WST-8, Boyden chamber, and cell-tracking assays. These findings suggest that nestin may at least partially mediate these behaviors of canine mammary carcinoma cells.

Expression and Roles of S100A4 in Anaplastic Cells of Canine Mammary Carcinomas.

S100A4 (metastasin), a member of the S100 protein family, was initially identified in metastatic cells and is well established as a marker of aggressive human cancer. However, expression and roles of S100A4 in canine mammary tumors have not been clarified. In this study, expression of S100A4 was examined immunohistochemically in normal, hyperplastic, and neoplastic mammary glands of dogs. In all normal and benign lesions, S100A4 was restricted to a few stromal fibroblasts and inflammatory cells. However, in 7 of 57 (12%) of the malignant tumors examined, cytoplasmic and nuclear expression of S100A4 was observed in epithelial tumor cells and stromal cells. Particularly, the frequency of S100A4-positive anaplastic carcinomas was high (4/8 cases, 50%). Next, we established a novel cell line, named NV-CML, from a S100A4-positive canine mammary carcinoma. The cultured NV-CML cells and the tumors that developed in the immunodeficient mice after subcutaneous injection of the cells maintained the immunophenotype of the original tumor, including S100A4 expression. Using this cell line, we examined the cellular functions of S100A4 using RNA interference. S100A4 expression level in NV-CML cells transfected with small interfering RNA (siRNA) targeting canine S100A4 (siS100A4) was reduced to about one-fifth of those with negative-control siRNA (siNeg). Cell proliferation in WST-8 assay and cell migration in Boyden chamber assay were significantly decreased in siS100A4-transfected cells compared with siNeg-transfected cells. These findings suggest that S100A4 may be related to progression of canine mammary carcinomas via its influence on cell growth and motility.

Reduced expression of the H19 long non-coding RNA inhibits pancreatic cancer metastasis.

H19 is an oncofetal RNA expressed in the developing embryo as well as in bladder, breast, gastric, pancreatic, hepatocellular, and prostate cancers. Recent studies have shown that H19 enhances cancer invasion and metastasis; however, its roles in cancer remain controversial. In the current study, H19 exhibited the second largest increase (82.4-fold) and represented the only non-protein coding gene among 11 genes identified that were elevated over 10-fold in lung-metastasis-derived pancreatic cancer cells compared with their parental cells using a mouse metastatic model. Subsequently, we further clarified the roles of H19 in pancreatic cancer growth and metastasis using in vitro and in vivo techniques. In situ hybridization showed that H19 was detected in 23 of 139 invasive ductal carcinomas (17%), and that H19 expression positively correlated with higher histological grades (P < 0.0001). Overexpression of H19 in PANC-1 pancreatic cancer cells induced higher motilities, whereas H19 inhibition using shRNA and siRNA showed opposite results; however, cell growth rates were not impacted. Intravenous injection of H19 shRNA vector-transfected PANC-1 cells yielded marked inhibition of metastasis in the liver and lungs of immunodeficient mice. These findings suggest that H19 has important roles in pancreatic cancer metastasis, and that inhibition of H19 represents a novel candidate for pancreatic cancer therapy.

Nestin phosphorylation at threonines 315 and 1299 correlates with proliferation and metastasis of human pancreatic cancer.

The neuroepithelial stem cell marker nestin is a cytoskeletal protein that regulates cell proliferation, invasion, and stemness in various tumors, including pancreatic tumors. In the present study, we examined the expression and roles of phosphorylated nestin in pancreatic cancer cells. Nestin phosphorylation at threonines 315 (Thr315) and 1299 (Thr1299) was observed during mitosis in human pancreatic cancer cells. Nestin phosphorylation was positively correlated with a cell proliferation marker, MIB-1 expression in human pancreatic cancer samples. Transfection of MIA PaCa-2 cells with nestin mutated at Thr315 and/or Thr1299 (to suppress phosphorylation) resulted in lower proliferation rates than those in control groups. Transfecting MIA PaCa-2 cells with wild-type nestin or with nestin mutated at Thr315 increased migration and invasion. In contrast, transfection with nestin mutated at both phosphorylation sites (Thr315 and Thr1299) did not enhance cell migration or invasion. In an intra-splenic xenograft experiment using MIA PaCa-2 cells, tumors expressing the nestin double mutant formed fewer liver metastases than tumors expressing wild-type nestin. Nestin phosphorylation at these two sites was decreased upon treatment with inhibitors for cyclin dependent kinases, AKT, and Aurora in PANC-1 cells, which express a high baseline level of phosphorylated nestin. These findings suggest that phosphorylation of nestin at Thr315 and/or Thr1299 affects cell proliferation, and inhibition of both phosphorylation sites suppresses invasion and metastasis of human pancreatic cancer. Inhibiting nestin phosphorylation at these two sites may represent a novel therapeutic strategy for pancreatic cancer.

TERATOMA OF THE OVARY IN A FREE-RANGING JAPANESE RACCOON DOG (NYCTEREUTES PROCYONOIDES VIVERRINUS).

A young adult, female, free-ranging Japanese raccoon dog ( Nyctereutes procyonoides viverrinus) with scabies infection was found dead as a result of traumatic injuries presumed to reflect vehicular trauma. Necropsy showed a large solid mass located on the left ovarian region, occupying a third of the abdominal cavity. Histologically, the mass contained complex tissues derived from three germinal layers, with areas of cuboidal or columnar epithelium, keratinized squamous epithelium, bone, cartilage, and adipose tissue. This paper presents the first morphologic description of ovarian teratoma in a raccoon dog.

Mitotic index and multipolar mitosis in routine histologic sections as prognostic markers of pancreatic cancers: A clinicopathological study.

OBJECTIVES: Pancreatic cancer is characterized by genomic complexity and chromosomal instability, and atypical mitotic figures are morphological features of this phenotype. In the present study, we determined the frequency and the clinicopathological and prognostic significance of mitotic figures in pancreatic cancers. METHODS: We surveyed the mitotic figures of the normal ductal epithelium, acinar cells, pancreatic intraepithelial neoplasias, and pancreatic cancers on hematoxylin-and-eosin-stained tissue specimens (n = 121). RESULTS: Pancreatic cancer cells showed significantly higher mitotic indices as compared with the ductal cells, acinar cells, and pancreatic intraepithelial neoplasias. Both normal and atypical mitosis were significantly elevated only in pancreatic cancers. In pancreatic cancers, approximately 30% of total mitosis was atypical including multipolar, lag-type, ring and asymmetrical mitosis, and anaphase bridges. The Kaplan-Meier curves in pancreatic cancers showed significant correlations between total mitosis and disease free survival. Furthermore, the cases with multipolar mitosis showed poorer prognosis than those without. Lymph node metastasis and multipolar mitosis were independent prognostic factors for overall survival of patients with pancreatic cancer. In addition, lymph node metastasis and total mitosis were independent factors for disease free survival. CONCLUSION: These findings suggest that routinely obtained pathological specimens, even small biopsy or cytological specimens, can provide valuable information concerning the prognosis of pancreatic cancers.

Nestin delineates pancreatic cancer stem cells in metastatic foci of NOD/Shi-scid IL2Rγ(null) (NOG) mice.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a high incidence of hepatic metastases, as well as occasional pulmonary metastases. To delineate the potential role of cancer stem cells (CSCs) in PDAC metastasis, human PDAC cells were injected into the spleen of mice. The characteristics and expression of markers associated with CSC and epithelial-mesenchymal transition (EMT) of metastatic cells that developed in the liver and lung were then compared with parental cells. The metastatic cells were polygonal, and larger than parental cells. Metastatic cells also exhibited decreased proliferation and increased adhesion to extracellular matrices, as well as enhanced migration and invasion in vitro and increased metastatic capacity in vivo. The CSC markers ALDH1A1, ABCG2, and nestin were expressed at high levels in metastatic cells and exhibited changes consistent with EMT (eg, decreased E-cadherin expression). Moreover, metastatic cells readily formed spheres in culture and exhibited an increased side population by flow analysis. Nestin and ABCG2 were also expressed at high levels in metastatic lesions from PDAC patients, and silencing nestin with shRNA in PDAC cells derived from lung metastases resulted in a marked decrease in the capacity of the cells to form spheres and to yield pulmonary or hepatic metastases. Thus, the metastatic potential of human PDAC cells correlates with CSCs and with EMT characteristics and is dependent on nestin expression.

Inhibition of fibroblast growth factor receptor 2 attenuates proliferation and invasion of pancreatic cancer.

The alternative splicing of the extracellular domain of fibroblast growth factor receptor (FGFR)-2 generates the IIIb and IIIc isoforms. Expression of FGFR-2 IIIb correlates with vascular endothelial growth factor-A (VEGF-A) expression and venous invasion of pancreatic ductal adenocarcinoma (PDAC). By contrast, FGFR-2 IIIc expression correlates with faster development of liver metastasis after surgery, and increased proliferation rates and invasion of the cancer. In this study, we analyzed the expression and roles of total FGFR-2 (both isoforms) to determine the effectiveness of FGFR-2-targeting therapy for PDAC. Immunohistochemically, FGFR-2 was highly expressed in 25/48 (52.1%) PDAC cases, and correlated with advanced stage cancer. In FISH analysis, FGFR2 was amplified in 3/7 PDAC cell lines. We stably transfected an FGFR-2 shRNA targeting the IIIb and IIIc isoforms into FGFR2-amplified PDAC cells. The proliferation rates, migration, and invasion of FGFR-2-shRNA-transfected cells were lower than those of control cells in vitro. In response to FGF-2, FGFR-2-shRNA-transfected cells showed decreased phosphorylation of ERK compared with control cells. The FGFR-2-shRNA-transfected cells also expressed lower levels of vascular endothelial growth factor-A than control cells, and formed smaller s.c. tumors in nude mice. These findings suggest that FGFR-2 is a therapeutic target for inhibition in PDAC.

Aortic squamous metaplasia in a patient with aortoesophageal fistula secondary to thoracic aortic aneurysm: an autopsy case.

Aortoesophageal fistula (AEF) is highly lethal. A 74-year-old man presented with hematemesis and consciousness loss. He had a long-term history of hypertension and gout. Computed tomography revealed an aneurysm of the distal descending thoracic aorta, which was treated by insertion of an aortic stent graft. After 24 days of stenting, endoscopic examination revealed an AEF. After 6 months of stenting, he died owing to mediastinitis. On autopsy, macroscopically, we found a 4 × 2.5-cm, oval, well-circumscribed AEF. We identified squamous epithelium in the area surrounding the AEF that covered the thoracic aorta inner cavity. Immunohistochemical analysis revealed that the squamous epithelium in the thoracic aorta was positive for p63 and 34ßE12. In conclusion, we encountered a long-term AEF case with aortic squamous metaplasia. To the best of our knowledge, human aortic metaplasia has never been reported. In the present case, aortic squamous metaplasia retained continuity with the esophageal squamous epithelium; therefore, the migration of the squamous epithelium through the AEF may have been induced by aortic erosion.

Antitumor effect of bevacizumab in a xenograft model of canine hemangiopericytoma.

Canine hemangiopericytoma (CHP) is characterized by frequent local recurrence and increased invasiveness. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in tumors. The aim of the present study was to investigate the effect of a single dose of bevacizumab on a xenograft model of CHP. VEGF protein was secreted from cultured CHP cells and interacted with bevacizumab. Bevacizumab treatment suppressed tumor growth by inhibiting tumor angiogenesis, whereas no significant differences were observed in the proliferation index and apoptosis rates of treated and untreated mice. Thus, bevacizumab had antitumor effects in a xenograft model of CHP.

Phosphorylation of Thr(1495) of nestin in a mouse model of cerebral ischemia and reperfusion damage.

Nestin, a class VI intermediate filament protein, is expressed by neuronal progenitor cells in the subventricular zone (SVZ). In the present study, we analyzed the nestin expression and phosphorylation levels in nerve cells in a mouse model of cerebral ischemia and reperfusion. C57BL/6 mice were subjected to three-vessel occlusion for 14 min, and were killed either 1 or 4 days after the procedure. The percentages of cells in the SVZ that were positive for nestin, Thr(1495)-phosphorylated nestin or Ki67 did not significantly differ between the ischemic reperfusion and sham groups. Conversely, in the striatum and cornu ammonis 2 (CA2) regions, the mice at 4 days after ischemic reperfusion showed significantly higher numbers and percentages of nerve cells that were positive for nestin, Thr(1495)-phosphorylated nestin and Ki67 compared to results from the other groups. To our knowledge, this is the first description of phosphorylated nestin expression in neural progenitor cells in the SVZ of adult mice. In this cerebral ischemia and reperfusion mouse model, cells positive for Thr(1495)-phosphorylated nestin were increased in the striatum and CA2 field of the hippocampus; suggesting that nestin phosphorylation may play an important role in mitotically active neuronal progenitor cells.

[A case of quadruple cancer of the liver, stomach, bladder, and ureter].

A 67-year-old man with bladder cancer who was treated with transurethral resection of bladder tumour(TUR-Bt)and chemotherapy at the age of 59 years was diagnosed as having urothelial cancer by biopsy 8 years later. Detailed examination revealed the presence of synchronous triple cancer, with hepatocellular cancer and gastric cancer. Subsequently, semi-total gastrectomy, partial hepatectomy(S6), radio frequency ablation(S5, S7), and cholecystectomy were performed. Histologically, the gastric tumor was a moderately differentiated tubular adenocarcinoma, the hepatic tumor was a moderately differentiated hepatocellular carcinoma, the bladder tumor was a transitional cell carcinoma, and the ureteral tumor was an urothelial carcinoma.

Apocrine carcinoma-and-malignant myoepithelioma in a dog: a case of simultaneous malignant progression of both luminal epithelium and myoepithelium.

A 9-y-old male Boxer dog developed a mandibular skin tumor, which histologically had a locally invasive growth pattern composed of bilayered structures of inner eosinophilic cuboidal tumor cells and outer clear polygonal tumor cells with cytoplasm containing glycogen granules. Both cell populations gradually changed from low-grade morphologic features to highly anaplastic ones. Immunohistochemically, the eosinophilic tumor cells were positive for cytokeratin 8, a useful marker for luminal epithelial cells. In contrast, the clear tumor cells expressed several myoepithelial markers, including α-smooth muscle actin, p63, and cytokeratin 14. Based on these histologic and immunohistochemical characteristics, we diagnosed this apocrine sweat gland tumor as a carcinoma-and-malignant myoepithelioma with high-grade transformation of both luminal and myoepithelial cells. Our case may be a helpful reference for the histogenesis of carcinoma-and-malignant myoepithelioma, in which both the luminal epithelial and myoepithelial components are malignant.

Infective endocarditis with systemic bacterial embolism caused by Staphylococcus aureus in a free-ranging Amami rabbit (Pentalagus furnessi).

The Amami rabbit (Pentalagus furnessi) is found only on the two islands of Amami-Oshima and Tokunoshima in southwest Japan. It has a primitive appearance and ecology, is an evolutionarily valuable animal and has been assigned to the International Union for Conservation of Nature Red List of Threatened Species. We describe a case with mild purulent wounds on the distal digital skin of both forelimbs and multiple nodular lesions in various organs, including the heart and kidney. Microscopically, the heart lesions were characterized by disruption of the mitral valve and multifocal myocardial necrosis and abscesses due to infection with gram-positive cocci. Similar bacterial infarctions were also found in other organs, including the kidneys. The bacteria were identified as Staphylococcus aureus by immunohistochemical and molecular biological examinations. This first report of infective endocarditis and systemic infarctions caused by S. aureus in an Amami rabbit indicates the importance of monitoring purulent injuries, even if mild, to prevent secondary infections in this species.

Histological, immunohistochemical and ultrastructural features of polyglucosan bodies in uterine smooth muscle of pet rabbits (Oryctolaguscuniculus).

We document the frequency and morphological and immunohistochemical features of inclusion bodies in uterine smooth muscle cells in 56 (76%) of 74 investigated pet rabbits (Oryctolagus cuniculus). Inclusion bodies began to appear at the age of 2 years and their frequency increased with age (P = 0.047, r = 0.33). They ranged from 5 to 20 µm in diameter, were slightly basophilic to amphophilic with well-delimited oval bodies in haematoxylin and eosin-stained tissue sections and formed in the cytoplasm of the uterine smooth muscle cells with displacement of the cell nuclei. The inclusion bodies were positive with periodic acid-Schiff, Best's carmine, Lugol's iodine and Grocott's methenamine silver methods. They were immunoreactive to a monoclonal antibody raised against human polyglucosan and negative with monoclonal antibodies for several intermediate filament proteins. Electron microscopy revealed that they were non-membranous structures composed of electron-dense amorphous material. The morphological, histochemical, immunohistochemical and ultrastructural features of the inclusion bodies in the rabbi uteri were similar to those of human polyglucosan bodies (PGBs). PGBs appear to occur at a high frequency in the uterus of rabbits, which are known to be susceptible to uterine diseases.

Extra-gastrointestinal stromal tumor of the pelvic cavity: case report.

Extra-gastrointestinal stromal tumors (E-GISTs) not associated with the alimentary tract in the pelvic cavity are extremely rare. We treated a 49-year-old Japanese man with such an E-GIST in the pelvic cavity who underwent an intrapelvic tumorectomy with a total prostatectomy and partial rectum resection. Gross examination of the specimen revealed an 8.1 × 5 × 4 cm white-grayish mass. Histological findings showed uniform spindle cells with scant atypia that formed interlacing bundles or whorl patterns. These neoplastic cells did not invade adjacent organs, including the gut. Immunohistochemical findings revealed that the neoplastic cells were positive for c-kit, CD34, and vimentin. Molecular analysis showed a c-kit mutation at exon 9 with duplication of Ala and Tyr. Our diagnosis was E-GIST, which belongs to the intermediate group of GIST. Following the operation, we administered imatinib mesylate for 6 months. After stopping for 5 months, it was administered again for local recurrence. We are planning our future strategy for this case including surgical resection as necessary.

Gp130-Mediated STAT3 Activation Contributes to the Aggressiveness of Pancreatic Cancer through H19 Long Non-Coding RNA Expression.

Signaling pathways involving signal transducer and activator of transcription 3 (STAT3) play key roles in the aggressiveness of pancreatic ductal adenocarcinoma (PDAC), including their tumorigenesis, invasion, and metastasis. Cancer stem cells (CSCs) have been correlated with PDAC aggressiveness, and activation of STAT3 is involved in the regulation of CSC properties. Here, we investigated the involvement of interleukin-6 (IL-6) or the leukemia inhibitory factor (LIF)/glycoprotein 130 (gp130)/STAT3 pathway and their role in pancreatic CSCs. In PDAC CSC-like cells formed by culturing on a low attachment plate, autocrine/paracrine IL-6 or LIF contributes to gp130/STAT3 pathway activation. Using a gp130 inhibitor, we determined that the gp130/STAT3 pathway contributes to the maintenance of stemness features, the expression of membrane-type 1 matrix metalloproteinase (MT1-MMP), and the invasion of PDAC CSC-like cells. The gp130/STAT3 pathway also modulates the transforming growth factor (TGF)-ß1/Smad pathway required for epithelial-mesenchymal transition induction through regulation of TGFß-RII expression in PDAC CSC-like cells. Furthermore, chromatin immunoprecipitation assays revealed that p-STAT3 can access the active promoter region of H19 to influence this metastasis-related long non-coding RNA and contribute to its transcription in PDAC CSC-like cells. Therefore, the autocrine/paracrine IL-6 or LIF/gp130/STAT3 pathway in PDAC CSC-like cells may eventually facilitate invasion and metastasis, two hallmarks of malignancy. We propose that inhibition of the gp130/STAT3 pathway provides a promising strategy for targeting CSCs for the treatment of PDAC.

Three new species of Eimeria (Apicomplexa: Eimeriidae) from the Amami rabbit, Pentalagus furnessi (Mammalia: Leporidae).

The Amami rabbit, Pentalagus furnessi (Mammalia: Lagomorpha: Leporidae), is a relict and endangered species endemic to the Amami-Oshima and Tokunoshima Islands, located in southwestern Japan. Here, we described three new species of Eimeria (Apicomplexa: Eimeriidae) parasites detected from fecal samples of wild Amami rabbits. Eimeria furnessi n. sp., recorded in 21 (58.3%) samples, has ellipsoidal oocysts with two walls and micropyle, 26.0 × 16.6 µm, and elongate-ovoidal sporocysts, 13.1 × 6.3 µm, with Stieda body. Eimeria hilleri n. sp., recorded in 9 (25.0%) samples, has ellipsoidal oocysts with two walls and micropyle, 34.7 × 21.4 µm, and elongate-fusiform to elongate-ovoidal sporocysts, 15.7 × 8.3 µm, with Stieda and substieda bodies. Eimeria sagentae n. sp., recorded in 13 (36.1%) samples, has ellipsoidal oocysts with two walls and micropyle, 20.9 × 14.5 µm, and elongate-ovoidal sporocysts, 10.4 × 5.0 µm, with Stieda body. The three new species can be distinguished by the size and color of their oocysts. Further studies related to the pathogenicity of these parasites can improve the breeding and propagation procedures of the Amami rabbit.

Adenomatous Hyperplasia of Bowman's Capsule Epithelium in a Dog with Metastatic Nasal and Hepatic Neuroendocrine Carcinoma.

A 12-year-old spayed Shiba dog with a nasal neuroendocrine carcinoma and multiple hepatic nodules was necropsied. Histologically, proliferated blast cells with a monolayer or multilayered structure were observed in the kidney. This blast cell proliferation extended from Bowman's capsule epithelium to the proximal tubule in approximately 3% of nephrons. Immunohistochemistry revealed that blast cells were positive for vimentin, Wilm's tumour protein 1 (WT1), paired box 2 (PAX2) and CD10, but negative for cytokeratin (CK) AE1/AE3, CK19, CAM5.2, synaptophysin and chromogranin A. On the basis of these findings, adenomatous hyperplasia of Bowman's capsule epithelium was diagnosed. Multiple yellowish‒white nodules (1-3 cm) were found in the liver and diagnosed as neuroendocrine carcinoma with metastases to the lungs, adrenal glands and pancreaticoduodenal lymph nodes.

Multiple cystic sphere formation from PK-8 cells in three-dimensional culture.

Three-dimensional (3D) culture of cancer cells mimics the in vivo environment. Recently, we reported that pancreatic ductal adenocarcinoma (PDAC) cell lines with epithelial and mesenchymal features formed differently shaped spheres in 3D culture. However, only PK-8 cells, the epithelial PDAC cell line with the highest E-cadherin expression among the eight PDAC cell lines, formed multiple cystic spheres in 3D culture. Optical coherence tomography revealed interconnected cysts inside the spheres. A weak inter-cellular adhesion, individual cell degeneration, necrosis, and secretory granules in the cytoplasm were observed in the PK-8 spheres using electron microscopy. The expression of MUC1, MUC5AC, and amylase was increased in PK-8 cells in the 3D culture compared with that in 2D culture. These findings suggest that highly E-cadherin-expressing epithelial PK-8 cells form multiple cystic spheres, which may be promoted by enhanced mucin and amylase synthesis in 3D culture.