Artificial intelligence-based analysis of time-lapse images of sphere formation and process of plate adhesion and spread of pancreatic cancer cells.

Background: Most pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). Spherical morphology formed in three-dimensional (3D) cultures and the effects of anticancer drugs differ between epithelial and mesenchymal PDAC cell lines. In the human pancreas, cancer cells form 3D tumors, migrate to adjacent tissues, and metastasize to other organs. However, no effective methods exist to examine the ability of the tumor mass to migrate to surrounding tissues in vitro. We used spheres formed in 3D culture to investigate whether the migratory ability of tumors of PDAC cell lines, including epithelial and mesenchymal cell lines, varies. Methods: Sphere formation and adhesion and spread on culture plates were examined by artificial intelligence-based analysis of time-lapse imaging using five epithelial and three mesenchymal PDAC cell lines. Fused and non-fused areas of the sphere surface during sphere formation on low-attachment plates, the adhesion area to normal culture plates, and the sphere area maintaining its original form during adhesion to plates were measured. Results: Immunocytochemical staining confirmed that E-cadherin was highly expressed in epithelial PDAC spheres, as was vimentin in mesenchymal PDAC spheres, in 2D culture. When forming spheres using low-attachment plates, most epithelial PDAC cell lines initially showed decreased sphere area, and then the covering cells fused to form a smooth surface on the sphere. Mesenchymal PANC-1 and MIA PaCa-2 cells showed little reduction in sphere area and few areas of sphere surface fusion. When formed PDAC spheres were seeded onto normal culture plates, spheres of epithelial PK-8 cells-which have the highest E-cadherin expression, form numerous cysts, and have smooth sphere surfaces-did not adhere to normal plates even after 60 h, and epithelial PK45-P and T3M-4 spheres hardly adhered. Conversely, the area of adhesion and spread of mesenchymal PANC-1 and KP4 cell spheres on normal plates markedly increased from early on, forming large areas of attachment to plates. Conclusion: Seeding spheres formed in 3D culture onto culture plates can clarify differences in tumor migration potential to surrounding areas. The masses formed by each PDAC cell line varied in migratory ability, with mesenchymal PDAC masses being more migratory than epithelial PDAC masses.

Accelerated telomere shortening in adrenal zona reticularis in patients with prolonged critical illness.

Background: The number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells. Methods: Adrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis. Results: RTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women). Conclusions: This study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Single-cell RNA sequencing to detect age-associated genes that identify senescent cells in the liver of aged mice.

Senescent cells are predicted to occur and increase in animal tissues with aging. However, senescent cells in the tissues of aged animals remain to be identified. We refer to the marker genes to identify senescent cells in tissues as "age-associated genes". In this study, we searched for age-associated genes to identify senescent cells in the livers of aged animals. We performed single-cell RNA sequencing (scRNA-seq) to screen candidates for age-associated genes using young and aged rat primary hepatocytes. To remove animal species specificity, gene expression analyses in mouse livers were performed, confirming age-associated increases in the mRNA expression levels of Glipr1, Clec12a, and Phlda3. Moreover, the mRNA expression levels of Glipr1 and Phlda3 were increased by stress-induced premature senescence using doxorubicin in primary hepatocytes and livers of young mice. Transcriptome data of aged rat hepatocytes suggested that Glipr1, Clec12a, and Phlda3 were expressed in almost identical cells. Fluorescence in situ hybridization (FISH) confirmed the presence of cells with abundant Glipr1, Clec12a, and Phlda3 mRNA in 27-month-old mouse primary hepatocytes, which are considered to be senescent cells. This study is the first to identify Glipr1, Clec12a, and Phlda3 as age-associated genes in the mouse liver.

Clinicopathological and molecular characteristics of gastric papillary adenocarcinoma.

Papillary adenocarcinoma is defined as carcinoma with a well-defined papillary or villous structure. Despite sharing clinicopathological and morphological features with tubular adenocarcinomas, papillary adenocarcinomas frequently show microsatellite instability. The present study aimed to clarify the clinicopathological features, molecular classification, and programmed death-ligand 1 (PD-L1) expression characteristics of papillary adenocarcinoma, especially tumors with microsatellite instability. We examined the microsatellite status and expression of mucin core proteins and PD-L1 as well as the clinicopathological features in 40 gastric papillary adenocarcinomas. Surrogate immunohistochemical analysis of p53 and mismatch repair proteins along with Epstein-Barr virus-encoded RNA in situ hybridization were performed for molecular classification. Female predominance and frequent microsatellite instability were observed in papillary adenocarcinoma in comparison with tubular adenocarcinoma. The presence of microsatellite instability in papillary adenocarcinoma was significantly correlated with older age, tumor-infiltrating lymphocytes, and Crohn's-like lymphoid reactions. Surrogate examination demonstrated that the genomically stable type (17 cases, 42.5%) was the most common, followed by the microsatellite-unstable type (14 cases, 35%). Among the seven cases showing PD-L1-positive expression in tumor cells, four involved carcinomas with microsatellite instability. These results reveal the clinicopathological and molecular characteristics of gastric papillary adenocarcinoma.

Cardiac mucosa in neonates.

BACKGROUND: The histology of the cardiac mucosa at the esophagogastric junction (EGJ) at birth is still a controversy. We conducted a histopathological study of the EGJ to clarify the morphology, and to determine the presence or absence of cardiac mucosa at birth. SUBJECTS: We examined 43 Japanese neonates and infants that are born prematurely or at full term. Death had occurred between 1 and 231 days after birth. RESULTS: Cardiac mucosa without parietal cells showing positivity for anti-proton pump antibody, adjacent to the most distal squamous epithelium, was observed in 32 (74%) of the 43cases. Such mucosa was evident in neonates that were full-term and had died within 14 days after birth. On the other hand, cardiac mucosa with parietal cells adjacent to squamous epithelium was noted in 10 cases (23%); the remaining one (2%) had columnar-lined esophagus. Squamous and columnar islands were observed in a single histological section from the EGJ in 22 (51%) of the 43 cases. Parietal cells were sparsely or densely present in the gastric antral mucosa. CONCLUSIONS: On the basis of these histological findings, we consider that cardiac mucosa exists in neonates and infants and can be defined as such, irrespective of the presence or absence of parietal cells (so-called oxyntocardiac mucosa). Neonates born prematurely or at full-term have cardiac mucosa in the EGJ just after birth, as is the case for Caucasian neonates.

mTOR pathway as a potential therapeutic target for cancer stem cells in canine mammary carcinoma.

Mammary adenocarcinoma, the most common cancer in female dogs, often exhibits the lymph node and lung metastases and has a higher mortality rate. However, mammary adenocarcinoma has no established treatment, except early surgical excision. Canine mammary carcinoma has many common features with human mammary carcinoma, including clinical characteristics, heterogeneity, and genetic aberrations, making it an excellent spontaneous tumor model for human breast cancer. Diverse cancers comprised heterogeneous cell populations originating from cancer stem cells (CSCs) with self-renewal ability. Therefore, in addition to conventional therapy, therapeutic strategies targeting CSCs are essential for cancer eradication. The present study aimed to extract inhibitors of canine mammary CSCs that suppress their self-renewal ability. Sphere-formation assay, which evaluates self-renewal ability, was performed for the canine mammary cancer cell lines CTBp and CNMp. The spheres formed in this assay were used in inhibitor library screening, which identified various signaling pathways such as proteosome, stress inducer, and mammalian target of rapamycin (mTOR). The present study focused on the mTOR signaling pathway. Western blotting showed higher levels of phosphorylated mTOR in sphere-forming CTBp and CNMp cells than in adherent cells. Drug sensitivity examination using the mTOR inhibitors everolimus and temsirolimus revealed dose-dependent reductions in viability among both sphere-forming cells and adherent cells. Expression of phosphorylated mTOR in adherent and sphere-forming cells decreased by everolimus and temsirolimus treatment. In mice transplanted with CTBp-derived spheres, everolimus treatment significantly decreased tumor volume compared to control. These results reveal that the mTOR signaling pathway may be a potential to be a therapeutic target in both cancer cells and CSCs. Novel therapeutic strategies for canine mammary carcinoma are expected to benefit to human breast carcinoma as well.

Spatiotemporal changes of tissue glycans depending on localization in cardiac aging.

Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells. METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

Mechanistic insights into cancer drug resistance through optogenetic PI3K signaling hyperactivation.

Hyperactivation of phosphatidylinositol 3-kinase (PI3K) signaling is a prominent feature in cancer cells. However, the mechanism underlying malignant behaviors in the state remains unknown. Here, we describe a mechanism of cancer drug resistance through the protein synthesis pathway, downstream of PI3K signaling. An optogenetic tool (named PPAP2) controlling PI3K signaling was developed. Melanoma cells stably expressing PPAP2 (A375-PPAP2) acquired resistance to a cancer drug in the hyperactivation state. Proteome analyses revealed that expression of the antiapoptotic factor tumor necrosis factor alpha-induced protein 8 (TNFAIP8) was upregulated. TNFAIP8 upregulation was mediated by protein translation from preexisting mRNA. These results suggest that cancer cells escape death via upregulation of TNFAIP8 expression from preexisting mRNA even though alkylating cancer drugs damage DNA.

Telomere Attrition in Intraductal Papillary Mucinous Neoplasms of the Pancreas Associated With Carcinogenesis and Aging.

OBJECTIVES: It is challenging to preoperatively distinguish malignant and benign forms of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. The aims of this study were to investigate whether telomere length is associated with pathological grade of IPMNs and age and to clarify the utility of telomere length as a marker to identify malignant IPMNs. METHODS: Pancreas tissue was obtained from 28 patients after resection. We measured the telomere lengths of tumor cells in IPMNs and normal duct cells by quantitative fluorescence in situ hybridization. The association of normalized telomere-centromere ratio (NTCR) to pathological grade of IPMNs and age were determined. RESULTS: The NTCR showed a gradual decrease with increasing pathological grade of IPMNs. The NTCR in intermediate- and high-grade dysplasia and adenocarcinoma lesions was significantly shorter than in normal pancreatic ducts (P < 0.05). In multivariate analysis, telomere length was most associated with carcinogenesis. When the cutoff value of NTCR was set to 0.74, the sensitivity for detection of high-grade dysplasia and adenocarcinoma was 82.8%, with a specificity of 87.5%. CONCLUSIONS: Telomere shortening occurs with carcinogenesis and aging. A significant reduction of telomere length in IPMNs may be useful for surgical decision making.

PRC2-dependent regulation of ganglioside expression during dedifferentiation contributes to the proliferation and migration of vascular smooth muscle cells.

Phenotypic switching between contractile (differentiated state) and proliferative (dedifferentiated state) vascular smooth muscle cells (VSMCs) is a hallmark of vascular remodeling that contributes to atherosclerotic diseases. Gangliosides, a group of glycosphingolipids, have been detected in atherosclerotic lesions and are suspected to contribute to the disease process. However, the underlying mechanism, specifically with respect to their role in VSMC phenotype switching, is not clear. In this study, we sought to reveal the endogenous expression of gangliosides and their functional significance in VSMCs during atherosclerosis. We found that switching from the contractile to proliferative phenotype was accompanied by upregulation of a- and b-series gangliosides, which in turn, were regulated by polycomb repressor complex 2 (PRC2). Downregulation of ganglioside expression using an siRNA targeting ST3GAL5, which is required for the synthesis of a- and b-series gangliosides, attenuated the proliferation and migration of dedifferentiated VSMCs. Therefore, we concluded that the increased expression of a- and b-series gangliosides via PRC2 activity during dedifferentiation is involved in the proliferation and migration of VSMCs. Gangliosides may be an effective target in VSMCs for atherosclerosis prevention and treatment.

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.

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.

Epithelial and Mesenchymal Features of Pancreatic Ductal Adenocarcinoma Cell Lines in Two- and Three-Dimensional Cultures.

Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer that is difficult to diagnose early, and there is no cure other than surgery. PDAC is classified as an adenocarcinoma that has limited effective anticancer drug and molecular-targeted therapies compared to adenocarcinoma found in other organs. A large number of cancer cell lines have been established from patients with PDAC that have different genetic abnormalities, including four driver genes; however, little is known about the differences in biological behaviors among these cell lines. Recent studies have shown that PDAC cell lines can be divided into epithelial and mesenchymal cell lines. In 3D cultures, morphological and functional differences between epithelial and mesenchymal PDAC cell lines were observed as well as the drug effects of different anticancer drugs. These effects included gemcitabine causing an increased growth inhibition of epithelial PDAC cells, while nab-paclitaxel caused greater mesenchymal PDAC cell inhibition. Thus, examining the characteristics of epithelial or mesenchymal PDAC cells with stromal cells using a 3D co-culture may lead to the development of new anticancer drugs.

Sphingomyelin localization in the intestinal crypt surface.

Macroscopic lipid observation in the organs of living small animals has not been realized. Here, we visualized sphingomyelin (SM) in the intestines of living mice using an SM-binding protein (EqtII-EGFP-His) under two-photon microscopy. The SM was identified as 10 µm spots in glands of the lamina propria of the mucosa in the large and small intestines. The spots vertically penetrated from the serosa toward the mucosal side. At the edge of the mucosal side in the small intestine, these spots connected with each other and formed horizontal lines. For the large intestine, the horizontal lines became a surface, indicating that SM covered the whole crypt membrane. Detailed observation revealed thin SM-positive lines that connected the spots and the blood vessels in the small intestine. Thus, SM exists at crypt surfaces and inside crypts of the intestines and can regulate the functions of the digestion system.

TGF-ß1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-ß1) and whether EMT could be induced through TGF-ß1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-ß1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-ß1. Analysis of EMT markers showed that mRNA levels changed with TGF-ß1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-ß1 treatment. Invasion assays showed that TGF-ß1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-ß1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.

A New Anorectal Melanoma Cell Line Derived from a Primary Human Rectal Tumor.

BACKGROUND: Anorectal melanoma is a rare disease with a poor prognosis. Symptoms are often nonspecific, which complicates preoperative diagnosis. Here, we describe the establishment of MELS, a new anorectal melanoma cell line derived from resection of a rectal tumor in a 40-year-old Japanese man. METHODS: Histological, electron microscopic, and immunohistochemical features of S-100, HMB-45, Melan-A, and NSE positivity of the tumor were typical of surgically resected anorectal melanoma. RESULTS: MELS cells are round or oval and have sharp thorn-like protrusions on some or all cell membranes. The cells form irregular attached colonies with numerous floating cells in two-dimensional culture. Transmission electron microscopy revealed that some MELS cells have cytoplasmic melanosomes. Immunocytochemically, MELS cells and surgical tissues had the same staining pattern. MELS cells had lower growth rates than Caco-2 (a colon adenocarcinoma cell line) and A375 (a cutaneous melanoma cell line) cells. Oxaliplatin and irinotecan were more effective in MELS cells than in Caco-2 and A375 cells. CONCLUSIONS: No previous report provided detailed clinical information on an anorectal melanoma cell line. Thus, MELS cells should improve our understanding of the biological characteristics of anorectal melanoma and provide a novel platform for examining the effects of therapies for anorectal melanoma.

Telomere lengths in Barrett's esophagus as a precancerous lesion.

BACKGROUND: We have reported that precancerous conditions and lesions invariably have shorter telomeres and associated chromosomal instability relative to normal tissue. METHODS: Using the Q-FISH method and our original software, Tissue Telo, we estimated telomere lengths in cardiac- and intestinal-type mucosae in 48 cases of Barrett's esophagus (short-segment (SS) n = 18; long-segment (LS) n = 30). RESULTS: There were no significant differences in telomere length between the cardiac and intestinal types in any of the 48 cases, suggesting that the presence or absence of goblet cells in the columnar segments is unrelated to telomere-dependent chromosomal instability in Barrett's esophagus. In LS Barrett's esophagus, telomeres were shorter in cardiac-type than in intestinal-type mucosa, suggesting that the former may play a more important role than the latter as a precancerous lesion in LS. Telomeres in cardiac-type mucosa were longer in SS than in LS, supporting the possibility that cardiac-type LS may pose a higher risk as a precancerous lesion than cardiac-type SS. CONCLUSIONS: Although it has been considered that Barrett's carcinoma arises only from intestinal-type mucosa, our present findings support previous histogenetic studies suggesting that cardiac-type mucosa is more important as a precancerous condition in Barrett's esophagus than anticipated.

Gangliosides as Signaling Regulators in Cancer.

At the plasma membrane, gangliosides, a group of glycosphingolipids, are expressed along with glycosphingolipids, phospholipids, and cholesterol in so-called lipid rafts that interact with signaling receptors and related molecules. Most cancers present abnormalities in the intracellular signal transduction system involved in tumor growth, invasion, and metastasis. To date, the roles of gangliosides as regulators of signal transduction have been reported in several cancer types. Gangliosides can be expressed by the exogenous ganglioside addition, with their endogenous expression regulated at the enzymatic level by targeting specific glycosyltransferases. Accordingly, the relationship between changes in the composition of cell surface gangliosides and signal transduction has been investigated by controlling ganglioside expression. In cancer cells, several types of signaling molecules are positively or negatively regulated by ganglioside expression levels, promoting malignant properties. Moreover, antibodies against gangliosides have been shown to possess cytotoxic effects on ganglioside-expressing cancer cells. In the present review, we highlight the involvement of gangliosides in the regulation of cancer cell signaling, and we explore possible therapies targeting ganglioside-expressing cancer.

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.