Single-cell analysis reveals the transcriptional alterations in the submandibular glands of aged mice.

OBJECTIVES: Aging-related salivary gland changes, such as lymphocyte infiltration and acinar cell loss decrease saliva secretion, thereby affecting quality of life. The precise molecular mechanisms underlying these changes remain unclear. METHODS: We here performed single-cell RNA sequencing to clarify gene expression changes in each cell type comprising the submandibular glands (SMGs) of adult and aged mice. RESULTS: The proportion of acinar cells decreased in various epithelial clusters annotated with cell type-specific marker genes. Expression levels of the cellular senescence markers, Cdkn2a/p16 and Cdkn1a/p21, were increased in the basal and striated ducts of aged SMGs relative to their levels in those of adult SMGs. In contrast, senescence-associated secretory phenotype-related genes, except transforming growth factor-ß, exhibited little change in expression in aged SMGs relative to adult SMGs. CONCLUSIONS: Gene Ontology analysis revealed increased expression levels of genes encoding major histocompatibility complex (MHC) class I components in the ductal component cells of aged SMGs. MHC class I expression may thus be associated with salivary gland aging.

BMP-2-mediated signaling suppresses salivary gland development.

Research regarding the process of salivary gland development and elucidation of related mechanisms are considered essential for development of effective treatments for conditions associated with salivary disease. Various reports regarding the effects of bone morphogenetic protein (BMP)-2 on hard tissue cells have been presented, though few have examined those related to salivary gland formation. Using an organ culture system, the present study was conducted to investigate the function of BMP-2 in salivary gland formation. Salivary glands obtained from embryonic day 13.5 mice and treated with BMP-2 showed suppression of primordial cell differentiation and also gland formation in a concentration-dependent manner. Furthermore, gland formation inhibition was suppressed by concurrent treatment with dorsomorphin, an inhibitor of the Smad pathway. Expression levels of AQP5, a marker gene for acinar cells, and Prol1, an opiorphin expressed in the lacrimal gland, were decreased in salivary glands treated with BMP-2. The present findings indicate that suppression of salivary gland formation, especially acinar differentiation, is induced by BMP-2, a phenomenon considered to be related to the Smad pathway.

Role of Snai2 and Notch signaling in salivary gland myoepithelial cell fate.

Myoepithelial (ME) cells in exocrine glands exhibit both epithelial and mesenchymal features, contributing to fluid secretion through contraction. However, the regulation mechanism of behind this unique phenotype in salivary glands remains unclear. We established a flow cytometry-based purification method using cell surface molecules, epithelial cell adhesion molecule (EpCAM) and alpha 6 integrin (CD49f), to characterize ME cells. EpCAM+CD49fhigh cells showed relatively high expression of ME cell-marker genes, such as alpha-smooth muscle actin (α-SMA). For lineage tracing and strict isolation, tdTomato+EpCAM+CD49fhigh-ME cells were obtained from myosin heavy chain 11 (Myh11) -CreERT2/tdTomato mice. Transcriptome analysis revealed that expression of genes involved in the epithelial-mesenchymal transition, including Snai2, were upregulated in the ME cell-enriched subset. Snai2 suppression in stable ME cells decreased α-SMA and increased Krt14 expression, suggesting that ME cell features may be controlled by the epithelial-mesenchymal balance regulated by Snai2. In contrast, ME cells showed reduced ME properties and expressed the ductal markers Krt18/19 under sphere culture conditions. Notch signaling was activated under sphere culture conditions; excessive activation of Notch signaling accelerated Krt18/19 expression, but reduced α-SMA and Snai2 expression, suggesting that the behavior of Snai2-expressing ME cells may be controlled by Notch signaling.

Synovial sarcoma of the stomach: a case report and a systematic review of literature.

Worldwide, 5-10% of soft tissue sarcoma cases in adults have been attributed to synovial sarcoma. It is often reported to occur near the joints of the arm, neck, and leg but rarely in the gastrointestinal tract. In this study, we report a case of synovial sarcoma arising in the stomach of a 59-year-old woman. Gastrointestinal endoscopy revealed an ulcerative and hemorrhagic tumor with marginal elevation in the fundus. Histological study showed that the tumor was composed of tightly packed spindle cells in bundles, and one of its component demonstrated significant mitotic activity (> 40/10 high-power fields) in several areas. The diagnosis was confirmed by the evidence of SS18 gene rearrangement, according to immunohistochemistry study, (including a novel SS18-SSX fusion-specific antibody), fluorescent in situ hybridization, and the identification of the SS18-SSX1 and SS18-SSX1/2/4 fusion transcripts using reverse-transcript polymerase chain reaction. No evidence of local recurrence or distant metastasis has been found in the more than 5 years since. Distinguishing synovial sarcoma in the digestive tract from other mesenchymal neoplasms, such as gastrointestinal stromal tumor, may be difficult, especially when spindle-shaped cell proliferation is predominant, as in our patient. Therefore, morphological, immunohistological, and molecular evaluations are important for a comprehensive diagnosis.

Sox9 function in salivary gland development.

BACKGROUND: Organogenesis is regulated by morphogen signaling and transcription networks. These networks differ between organs, and identifying the organ-specific network is important to clarify the molecular mechanisms of development and regeneration of organs. Several studies have been conducted to identify salivary gland-specific networks using a mouse submandibular gland model. The submandibular glands (SMGs) of mice manifest as a thickening of the oral epithelium at embryonic day 11.5 and invaginate into the underlying mesenchyme. The network between Fgf10 and Sox9 is involved in SMG development in mice. HIGHLIGHT: Sox9, a member of the Sox family, is expressed in the SMG in mice from the embryonic stage to the adult stage, although the distribution changes during development. A null mutation of mouse Sox9 is lethal during the neonatal period due to respiratory failure, whereas deletion of Sox9 in the oral epithelium using the Cre/lox P system, can lead to smaller initial buds of SMGs in conditional knockout (cKO) mice than in normal mice. In addition, we showed that adenoviral transduction of Sox9 and Foxc1 genes into mouse embryonic stem cell-derived oral ectoderm could induce salivary gland rudiment in an organoid culture system. ChIP-sequencing revealed that Sox9 possibly regulates several tube- and branching-formation-related genes. CONCLUSION: Sox9 may serve as an essential transcription factor for salivary gland development. The Sox9-mediated pathway can be a promising candidate for regenerating damaged salivary glands.

Genetic and histopathological analysis of a case of primary intraosseous carcinoma, NOS with features of both ameloblastic carcinoma and squamous cell carcinoma.

BACKGROUND: Primary intraosseous carcinoma (PIOC), NOS is an odontogenic carcinoma with unknown etiology. Its diagnosis may be used when central jaw carcinoma cannot be categorized as any other type of carcinoma. Further information on this extremely rare tumor is needed to improve our understanding and evaluate the classification of odontogenic carcinomas. CASE PRESENTATION: We herein presented two patients with PIOC, NOS with different clinical and histopathological features and analyzed gene mutations in these patients using next-generation sequencing (NGS). The typical PIOC, NOS case had many histopathological similarities to oral squamous cell carcinoma (OSCC), including the missense point mutations of TP53 Glu285Val, KDR Gln472His, and APC Pro1433Leu, which are similar to those in other cancers; however, no mutations were detected in the other patient with an atypical presentation of PIOC, NOS, which was derived from a precursor cystic lesion with similarities to both ameloblastic carcinoma and OSCC. CONCLUSIONS: Genetic analysis suggested that these two PIOC, NOS cases have different features and can be subcategorized.

Sox9 regulates the luminal stem/progenitor cell properties of salivary glands.

Exocrine glands share a common morphology consisting of ductal, acinar, and basal/myoepithelial cells, but their functions and mechanisms of homeostasis differ among tissues. Salivary glands are an example of exocrine glands, and they have been reported to contain multipotent stem cells that differentiate into other tissues. In this study, we purified the salivary gland stem/progenitor cells of adult mouse salivary glands using the cell surface marker CD133 by flow cytometry. CD133+ cells possessed stem cell capacity, and the transplantation of CD133+ cells into the submandibular gland reconstituted gland structures, including functional acinar. CD133+ cells were sparsely distributed in the intercalated and exocrine ducts and expressed Sox9 at higher levels than CD133- cells. Moreover, we demonstrated that Sox9 was required for the stem cell properties CD133+ cells, including colony and sphere formation. Thus, the Sox9-related signaling may control the regeneration salivary glands.

Alpha-L-fucosidase-1 is a diagnostic marker that distinguishes mucoepidermoid carcinoma from squamous cell carcinoma.

Alpha-L-fucose is a component of glycans on the cell surface. Alpha-L-fucose is correlated with tumorigenesis and malignancy, and alpha-L-fucosidase-1 (FUCA1), the enzyme that removes terminal α-L-fucose residues from glycoproteins, is downregulated in some high malignancy cancers. The expression profile of FUCA1 in head and neck tumors remains unknown, and we analyzed the expression profiles of FUCA1 and an upstream molecule p53 in mucoepidermoid carcinoma (MEC) and oral squamous cell carcinoma (OSCC). FUCA1 was expressed in most MECs irrespective of histopathological grading, whereas expression in OSCCs was low. High immunohistochemical intensity of p53 was detected in OSCCs at high frequency, but rarely detected in MECs. Genetic mutation analysis using next-generation sequencing revealed no significant mutation of TP53 in MECs. We further analyzed the expression profiles of FUCA1 in normal major and minor salivary glands and found strong expression in the intercalated duct, moderate expression in mucous acinar cells and no expression in serous acinar cells. These contrasting immunohistochemical profiles and anatomical distribution in normal salivary glands suggest that FUCA1 is a useful marker to distinguish MEC from OSCC, and many MECs have similar immunohistochemical phenotypes to intercalated duct and mucous acinar cells.

The glomus tumor resorbed bone and teeth in the mandible: a case report.

BACKGROUND: A glomus tumor is a rare neoplasm usually found in the dermis or subcutaneous tissue of the extremities. It is rare for the glomus tumor to occur on the head and face. Only 26 glomus tumors of the oral region and affected bone have been reported in the English-language literature (Table 1). We report a case of a glomus tumor at the mandible. As a new point, the glomus tumor resorbed a bone and teeth roots when the tumor progressed into the mandible. CASE PRESENTATION: The patient was a 44-year-old Japanese man who complained swelling of the right mandible. Radiographic examination showed a multilocular radiolucency area in the left mandible. Radiographic findings on our case resembled those of a common benign tumor. The lesion occupied to the premolar and molar area and revealed that the tumor resorbed the roots of the teeth. The lesion was removed surgically with the buccal cortical bone and buccal mucosa in contact with the mass of the tumor. The mass fully excised intraorally under general anesthesia, and the inferior alveolar nerve in contact with the mass was preserved. The specimen was pathologically diagnosed as a glomus tumor. Immunohistochemical staining was positive for vimentin, muscle-specific actin/HHF35, and calponin. A hairline-shaped area of positive staining for type IV collagen surrounding the tumor cells was also observed. In contrast, staining for alpha-SMA, cytokeratin (AE1/AE3), cytokeratin (CAM5.2), CK19, CD31, CD34, CD68, p63, S-100, Factor VIII, and desmin was all negative. The Ki-67 labeling index was almost 1%. A recurrent tumor was again detected in the site below the primary tumor at an 8-year follow-up, and it was surgically removed. The patient has had no symptoms of recurrence in 2 years after the second operation. CONCLUSION: The glomus tumor resorbed a bone and teeth roots when the tumor progressed into the mandible. The immunohistochemical features of the tumor were consistent with those described in previous reports. It is important to completely remove the Glomus tumor.

Oral carcinoma: Clinical evaluation using diffusion kurtosis imaging and its correlation with histopathologic findings.

PURPOSE: In this study, we aimed to determine the usefulness of diffusion kurtosis imaging (DKI) as a noninvasive method for evaluation of the histologic grade and lymph node metastasis in patients with oral carcinoma. MATERIALS AND METHODS: Twenty-seven patients with oral carcinoma were examined with a 3-T MR system and 16-channel coil. DKI data were obtained by a single-shot echo-planar imaging sequence with repetition time, 10,000 ms; echo time, 94 ms; field of view, 250 × 204.25 ms; matrix, 120 × 98; section thickness, 4 mm; four b values of 0, 500, 1000, and 2000 s/mm2; and motion-probing gradients in three orthogonal directions. Diffusivity (D) and kurtosis (K) were calculated using the equation: S = S0 ∙ exp(-b ∙ D + b2 ∙ D2 ∙ K/6). Conventional apparent diffusion coefficient (ADC) was also calculated. The MR images were compared with the histopathologic findings. RESULTS: Relative to the histologic grades (Grades 1, 2, and 3) of the 27 oral carcinomas, D values showed a significant inverse correlation (r = -0.885; P < 0.001) and K values showed a significant positive correlation (r = 0.869; P < 0.001), whereas ADC values showed no significant correlation (r = -0.311; P = 0.115). When comparing between metastatic and non-metastatic lymph nodes, significant differences in the D values (P < 0.001) and K values (P < 0.001), but not the ADC values (P = 0.110) became apparent. CONCLUSIONS: In patients with oral carcinoma, DKI seems to be clinically useful for the evaluation of histologic grades and lymph node metastasis.

A lesion categorized between ghost cell odontogenic carcinoma and dentinogenic ghost cell tumor with CTNNB1 mutation.

Ghost cell odontogenic carcinoma (GCOC) is a rare malignant neoplasm characterized by the presence of ghost cells. It is considered to arise either de novo or from a preexisting benign precursor, calcifying odontogenic cyst (COC), or dentinogenic ghost cell tumor (DGCT). We report a case of a 44-year-old Japanese male with a left maxillary tumor. The patient received treatment to resect the left maxillary cyst 25 years prior; however, the details were uncertain. The tumor was resected with clear margins. Taken together with the results of histological and immunohistochemical examinations, the tumor was categorized between GCOC and DGCT, and we diagnosed the tumor as GCOC suggesting similarity to DGCT. Further, we focused on CTNNB1, which encodes ß-catenin and is frequently mutated in COCs. In this tumor, we identified CTNNB1 Ser33Cys, one of the mutations typically found in COCs. This finding suggests that CTNNB1 is a common target for the pathogenesis of tumors accompanied by ghost cells.

Genetic basis of calcifying cystic odontogenic tumors.

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic tumors that form abnormally keratinized ghost cells. Mutations in CTNNB1, which encodes beta-catenin, have been implicated in the development of these tumors, but a causal relationship has not been definitively established. Thus, mutational hot spots in 50 cancer genes were examined by targeted next-generation sequencing in 11 samples of CCOT. Mutations in CTNNB1, but not in other genes, were observed in 10 of 11 cases. These mutations constitutively activate beta-catenin signaling by abolishing the phosphorylation sites Asp32, Ser33, or Ser37, and are similar to those reported in pilomatrixoma and adamantinomatous craniopharyngioma. In contrast, BRAF or NRAS mutations were observed in 12 and two control samples of ameloblastoma, respectively. In HEK293 cells, overexpression of mutated CTNNB1 also upregulated hair keratin, a marker of ghost cells. Furthermore, ghost cells were present in two cases of ameloblastoma with BRAF and CTNNB1 mutations, indicating that ghost cells form due to mutations in CTNNB1. The data suggest that mutations in CTNNB1 are the major driver mutations of CCOT, and that CCOT is the genetic analog of pilomatrixoma and adamantinomatous craniopharyngioma in odontogenic tissue.

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma.

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.

Differential expression of Toll-like receptors in follicular lymphoma, diffuse large B-cell lymphoma and peripheral T-cell lymphoma.

Although Toll-like receptors (TLRs) in mammals are well-known to play important roles in innate immunity, newer roles for the TLRs have suggested that cells with aberrant TLR expression may have a survival advantage over normal cells. Lymphocytes are one of a small number of cell types that express many of the TLRs, suggesting that abnormal TLR levels/signaling may potentially influence the progression of malignant lymphomas. Thus, frozen samples of 51 lymph nodes from patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and peripheral T-cell lymphoma (PTCL) were analyzed for the expression of TLR1 to 9 using quantitative real-time PCR, and compared to those in reactive lymphadenopathy (RL) samples. TLR2 was over-expressed in both DLBCL and PTCL but not in FL when compared to RL. TLR1 and TLR4 expression was up-regulated in PTCL, while TLR8 was highly expressed in DLBCL. Although TLR5 showed lower expression in FL, expression of TLR3, TLR6, TLR7 and TLR9 did not vary significantly between different lymphoma subtypes. Double immunostaining revealed an increase in the number of TLR2 and/or TLR8 expressing lymphoma cells in DLBCL. In PTCL, TLR2 and TLR4 expression was localized to neoplastic T cells. TLR expression is highly variable among lymphoma subtypes. However, despite this some significant differences exist that may prove useful in the development of novel therapeutic strategies.