An Autopsy Case of Myotonic Dystrophy Type 1 With Pancreatic Intraductal Papillary Mucinous Neoplasm.

Here, we present an autopsy case of long-standing myotonic dystrophy type 1 (DM1) in a patient who developed a pancreatic intraductal papillary mucinous neoplasm (IPMN). DM1 is a progressive genetic disorder that affects multiple organs, including the respiratory muscles. Several nationwide registry-based cohort studies have suggested that patients with DM1 have an increased risk of developing pancreatic cancers such as pancreatic ductal adenocarcinoma (PDAC). Pancreatic IPMNs are thought to progress from benign neoplasms to invasive cancers, and surgical specimens are usually required for the pathological diagnosis of pancreatic IPMNs. Although certain risk factors for developing pancreatic IPMNs reportedly overlap with those for PDAC, few cases of DM1 with pancreatic IPMNs have been reported. This is partly because pancreatectomy is associated with relatively high morbidity and mortality rates and few patients with DM1 who are suspected of having pancreatic IPMNs are candidates for surgical resection. Therefore, cases of DM1 with histopathologically diagnosed pancreatic IPMNs are rare, and the accumulation of such cases is important for understanding the association between DM1 and pancreatic IPMNs.

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.

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.

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.

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.

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.

Morphofunctional analysis of human pancreatic cancer cell lines in 2- and 3-dimensional cultures.

Genetic, transcriptional, and morphological differences have been reported in pancreatic ductal adenocarcinoma (PDAC) cases. We recently found that epithelial or mesenchymal features were enhanced in three-dimensional (3D) cultures compared to two-dimensional (2D) cultures. In this study, we examined the differences in the morphological and functional characteristics of eight PDAC cell lines in 2D and 3D cultures. Most PDAC cells showed similar pleomorphic morphologies in 2D culture. Under 3D culture, PDAC cells with high E-cadherin and low vimentin expression levels (epithelial) formed small round spheres encircled with flat lining cells, whereas those with high vimentin and low E-cadherin expression levels (mesenchymal) formed large grape-like spheres without lining cells and were highly proliferative. In 3D culture, gemcitabine was more effective for the spheres formed by PDAC cells with epithelial features, while abraxane was more effective on those with mesenchymal features. The expression levels of drug transporters were highest PDAC cells with high vimentin expression levels. These findings indicate that PDAC cells possess various levels of epithelial and mesenchymal characteristics. The 3D-culture method is useful for investigating the diversity of PDAC cell lines and may play important roles in the development of personalized early diagnostic methods and anticancer drugs for PDAC.

Polyvinyl alcohol increased growth, migration, invasion, and sphere size in the PK-8 pancreatic ductal adenocarcinoma cell line.

Polyvinyl alcohol (PVA) is a water-soluble synthetic polymer used in eye drops, embolization particles, and artificial cartilage. It has also been shown to cause expansion of functional multipotent self-renewing hematopoietic stem cells under serum-free conditions. In this study, we examined the effects of PVA on human pancreatic ductal adenocarcinoma (PDAC) cell lines using 2-dimensional (2D) and 3D-cultures with serum-free medium. In the 2D-culture, PVA-treatment induced an aggregated colony-like appearance in PDAC cells. It increased the growth of PK-8 cells in a dose-dependent manner as well as significantly increasing migration and invasion abilities. qRT-PCR showed an increase in α2 integrin and a decrease in matrix metalloprotease levels in PVA-treated PK-8 cells. Through qRT-PCR analysis, ß1 integrin expression at the mRNA level was found to be decreased; however, it was unaltered at the protein level when assessed using FACS analysis. PVA further induced mesenchymal to epithelial transition-like alterations, including increased E-cadherin and decreased Vimentin and N-cadherin expression. Four cancer stem cell (CSC) markers were higher in PVA-treated PK-8 cells compared to controls. In 3D-culture, PVA-treated PK-8 cells showed a rod-like appearance with larger sphere size and higher growth ability. qRT-PCR showed that CSC markers did not increase and 2 of 4 drug transporters had decreased in PVA-treated PK-8 cells. These findings suggest that PVA increases the growth, migration, invasion, and sphere size of PK-8 cells, but does not increase the proportion of pancreatic CSCs under 3D-culture conditions with serum-free medium.

Enhanced morphological and functional differences of pancreatic cancer with epithelial or mesenchymal characteristics in 3D culture.

Pancreatic cancer, composed of heterogeneous cancer cells, alters epithelial to mesenchymal features during growth and metastasis. In this study, we aimed to characterize pancreatic ductal adenocarcinoma (PDAC) cells showing epithelial or mesenchymal features in 3D culture. In 3D culture, PK-1 cells had high E-cadherin and low vimentin expression and exhibited a round-like appearance encircled by flat cells. PANC-1 cells had high vimentin and low E-cadherin expression and formed grape-like spheres. PK-1 cells had secretary granules and many microvilli, desmosomes, and adherens junctions, while PANC-1 cells had few microvilli, adherens junction, and no desmosomes. Cytokeratin 7, trypsin, CA19-9, and E-cadherin were highly expressed in PK-1 cells but not in PANC-1 cells. Ki-67 was diffusely expressed in PANC-1 spheres but was restricted to the peripheral flat cells of PK-1 spheres. PANC-1 and PK-1 cells were positive for transforming growth factor (TGF) ß receptor II and phosphorylated smad2/3, but PK-1 cells were smad4 negative. Taken together, 3D culture enhanced morphofunctional differences of PDAC cells showing epithelial or mesenchymal characteristics, and epithelial phenotype maintenance may be due to the ineffectiveness of the TGF- ß pathway. Clarification of heterogeneity using 3D culture may be useful for development of individualized diagnostic and therapeutic methods in patients with PDAC.