Functional transplantation of salivary gland cells differentiated from mouse early ES cells in vitro

Atrophy or hypofunction of the salivary gland because of aging or disease causes hyposalivation and has an effect on the quality of life of patients, for example not only dry mouth but deterioration in mastication/deglutition disorder and the status of oral hygiene. Currently conducted therapies for atrophy or hypofunction of the salivary gland in clinical practice are only symptomatic treatments with drugs and artificial saliva, and therefore it is preferable to establish a radical therapy. At this time, as a fundamental investigation, by co-culturing mouse early ES (mEES-6) cells with human salivary gland-derived fibroblasts (hSG-fibro), differentiation of mEES-6 cells to salivary gland cells has been attempted. Also, the possibility of cell engraftment was examined. After identifying the cells which were co-cultured with GFP-transfected mEES-6 cells and hSG-fibro, the cells were transplanted into the submandibular gland of SCID mice, and the degree of differentiation into tissues was examined. The possibility of tissue functional reconstitution from co-cultured cells in a three-dimensional culture system was examined. Our results confirmed that the co-cultured cells expressed salivary gland-related markers and had an ability to generate neo-tissues by transplantation in vivo. Moreover, the cells could reconstitute gland structures in a three-dimensional culture system. By co-culture with hSG-fibro, mEES-6 cells were successfully differentiated into salivary gland cells which were transplantable and have tissue neogenetic ability.     

Survival of salivary gland cancer stem cells requires mTOR signaling

Advanced salivary gland mucoepidermoid carcinoma (MEC) is a relentless cancer that exhibits resistance to conventional chemotherapy. As such, treatment for patients with advanced MEC is tipically radical surgery and radiotherapy. Facial disfigurement and poor quality of life are frequent treatment challenges, and many patients succumb to loco-regional recurrence and/or metastasis. We know that cancer stem-like cells (CSC) drive MEC tumorigenesis. The current study tests the hypothesis that MEC CSC are sensitive to therapeutic inhibition of mTOR. Here, we report a correlation between the long-term clinical outcomes of 17 MEC patients and the intratumoral expression of p-mTOR (p = 0.00294) and p-S6K1 (p = 0.00357). In vitro, we observed that MEC CSC exhibit constitutive activation of the mTOR signaling pathway (i.e., mTOR, AKT, and S6K1), unveiling a potential strategy for targeted ablation of these cells. Using a panel of inhibitors of the mTOR pathway, i.e., rapamycin and temsirolimus (mTOR inhibitors), buparlisib and LY294002 (AKT inhibitors), and PF4708671 (S6K1 inhibitor), we observed consistently dose-dependent decrease in the fraction of CSC, as well as inhibition of secondary sphere formation and self-renewal in three human MEC cell lines (UM-HMC-1,-3A,-3B). Notably, therapeutic inhibition of mTOR with rapamycin or temsirolimus induced preferential apoptosis of CSC, when compared to bulk tumor cells. In contrast, conventional chemotherapeutic drugs (cisplatin, paclitaxel) induced preferential apoptosis of bulk tumor cells and accumulation of CSC. In vivo, therapeutic inhibition of mTOR with temsirolimus caused ablation of CSC and downregulation of Bmi-1 expression (major inducer of stem cell self-renewal) in MEC xenografts. Transplantation of MEC cells genetically silenced for mTOR into immunodeficient mice corroborated the results obtained with temsirolimus. Collectively, these data demonstrated that mTOR signaling is required for CSC survival, and unveiled the therapeutic potential of targeting the mTOR pathway for elimination of highly tumorigenic cancer stem-like cells in salivary gland mucoepidermoid carcinoma.Subject terms: Cancer stem cells, Cancer stem cells, Head and neck cancer, Oral cancer     

Breast cancer cell-derived matrix supports vascular morphogenesis

The extracellular matrix (ECM), important for maintaining tissue homeostasis, is abnormally expressed in mammary tumors and additionally plays a crucial role in angiogenesis. We hypothesize that breast cancer cells (BCCs) deposit ECM that supports unique patterns of vascular morphogenesis of endothelial cells (ECs). Evaluation of ECM expression revealed that a nontumorigenic cell line (MCF10A), a tumorigenic cell line (MCF7), and a metastatic cell line (MDA-MB-231) express collagens I and IV, fibronectin, and laminin, with tenascin-C limited to MCF10A and MCF7. The amount of ECM deposited by BCCs was found to be higher in MCF10A compared with MCF7 and MDA231, with all ECM differing in their gross structure but similar in mean fiber diameter. Nonetheless, deposition of ECM from BCC lines was overall difficult to detect and insufficient to support capillary-like structure (CLS) formation of ECs. Therefore, a coculture approach was undertaken in which individual BCC lines were cocultured with fibroblasts. Variation in abundance of deposited ECM, deposition of ECM proteins, such as absent collagen I deposition from MDA231-fibroblast cocultures, and fibril organization was found. Deposited ECM from fibroblasts and each coculture supported rapid CLS formation of ECs. Evaluation of capillary properties revealed that CLS grown on ECM deposited from MDA231-fibroblast cocultures possessed significantly larger lumen diameters, occupied the greatest percentage of area, expressed the highest levels of von Willebrand factor, and expressed the greatest amount of E-selectin, which was upregulated independent of exposure to TNF-α. To our knowledge, this is the first study to report tumor cell ECM-mediated differences in vascular capillary features, and thus offers the framework for future investigations interrogating the role of the tumor ECM in supporting vascular morphogenesis.     

Potassium release from submandibular salivary gland in vitro

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K⁺ in response to α-adrenergic and muscarinic cholinergic stimulation. The system employed the specific α-, β-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists l-epinephrine and carbamylcholine both of which required the presence of Ca²⁺ for their effect. The introduction of Ca²⁺ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K⁺ release. Ouabain also allowed extensive K⁺ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K⁺ movement. K⁺ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca²⁺. The implication of cyclic GMP at some stage of K⁺ release was suggested by experiments with a phosphodiesterase inhibitor.The results support an hypothesis where the initial stimulus at either α-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca²⁺ enters the cells resulting in K⁺ release. The loss of K⁺ is quickly countered by the ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$) ATPase which would be activated by the lowered intracellular K⁺ levels.     

Cells of mouse submandibular salivary gland in culture in vitro

A cell culture that preserves its phenotype up to the 20th passage was obtained from mouse submandibular salivary glands. An analysis of the heterogeneous culture indicates the existence of several morphological types of cells, including small, densely packed cells of cuboidal or polygonal shapes and large, rounded cells. Epithelial cells of the submandibular gland cultured for several weeks were able to form tubular structures. Our studied cell culture of glandulocytes (cells of glandular epithelium) was represented by K19- and NGF-positive cells. It is important to note that, using both immunocytochemical staining and PCR, the expression of genes that encode the proinsulin and insulin proteins is revealed in the studied cell population.     

CD151 forms a functional complex with c-Met in human salivary gland cancer cells

In this study, we have attempted to elucidate the expression and function of CD151 in human salivary gland cancer cells. CD151 expression was detected in Acc2 and AccM cells, but not in normal tissues and primary cultured epithelial cells derived from human salivary gland. CD151 has been found to function as a molecular linker in the formation of complexes between c-Met/hepatocyte growth factor (HGF) receptor and integrin alpha3/alpha6. Knockdown of CD151 or integrin alpha3/alpha6 expression almost completely abrogated HGF-stimulated cell growth and migration. In contrast, forced expression of CD151 in Acc2 cells resulted in the increase of the HGF-dependent biological effects. These results suggest that CD151 forms a structural and functional complex with c-Met and integrin alpha3/alpha6, and exerts its oncogenic functions through excessive activation of the HGF/c-Met signalling pathway.     

Electron microscope studies on salivary gland cells

Summary A study of the ultrastructure of the nuclear envelope of the salivary glands cells in the sciarid Bradysia suggests that it probably consists of four membranes, instead of two as found in most cells. The fine structure associated with the pores closely resembles that described in amphibian oocytes by Wischnitzer 1958.British Empire Cancer Campaign.     

Early membrane injury in lethally irradiated salivary gland cells

The early manifestations of radiation injury in salivary glands were investigated in the rat. The animals received a single X-ray dose in the range of 200-2000 rad to their neck area. Glandular changes during the first 24 hours were studied by light and electron microscopy and by measuring serum amylase activity. The amount of cell necrosis was quantitated and expressed as necrosis index (NI), Parotid NI and serum amylase activity 24 hours following irradiation were directly proportional to the X-ray dose. The submandibular gland cells were radioresistant and so were the mucous cells of the sublingual gland. The major increase in parotid acinar cell necrosis occurred between 12 and 24 hours after irradiation. However, more than 100 per cent increase in serum amylase level was detected prior to the onset of any significant cell necrosis. As early as two hours following irradiation signs of cell membrane injury were demonstrable in the parotid by electron microscopy and consisted of intracellular oedema, sequestered degenerative cell membranes, and an accumulation of intramitochondrial particles. None of these changes was detectable in the submandibular gland. The implication of membrane injury in the lethal effects of radiation on parotid cells is discussed.     

Acetylcholine-induced responses in the salivary gland cells ofHelisoma trivolvis

This study describes the actions of acetylcholine (ACh) on the salivary gland cells of Helisoma. Perfusion of the salivary gland cells with ACh produces a long-lasting depolarization accompanied by an increase in the input conductance of the gland cells. The depolarization is often followed by a long-lasting hyperpolarization. Carbamylcholine, tetramethylammonium, and choline also produce depolarizing responses. Nicotine and pilocarpine produce only a small depolarization in the gland cells. The following cholinergic antagonists are effective in blocking the gland-cell response to ACh: tetraethylammonium, atropine, hexamethonium, d-tubocurarine, and strychnine. A new preparation, the "isolated acinus," was utilized to obtain the reversal potential of the ACh response. The mean reversal potential in 10 preparations was -7 +/- 8 mV. The depolarizing phase of the response is dependent on the presence of both external calcium and external sodium ions. The long-lasting hyperpolarization is produced by the activity of an electrogenic sodium-potassium pump. The properties of the acetylcholine receptors on the salivary gland cells of Helisoma are compared with those described in other gastropod preparations.     

Selection of malonate-resistant stromal cell-derived osteoprogenitor cells in vitro

Bone marrow stromal cells give rise to osteoprogenitor cell (OPC) colonies, with characteristic mineralized bone nodules in vitro. During differentiation, OPCs in the culture are surrounded by heterogeneous populations of various cell lineages and by different OPC differentiation stages. In the present study, attempts were made to increase the homogeneity of OPCs in culture. The reliance on energy metabolism restricted to glycolysis, which is specific to the premineralizing skeletal cells, was tested as a selectable marker for cells in this stage. Day 12 alkaline phosphatase (ALP) and day 20–21 calcium precipitates were used as early and late OPC differentiation markers. Malonate, a competitive inhibitor of succinate dehydrogenase, was added to the OPC stimulation medium, to interfere with the Krebs cycle-dependent energy metabolism operating in most of the stromal cells. OPCs that entered the stage of energy metabolism restricted to glycolysis were expected to become malonate resistant. Malonate showed dose and time dependence, 10 mM malonate added on day 3, decreased day 12 ALP activity/well to the lowest level. Variations in time and length of exposure to malonate used during the first 12 days of differentiation showed an inverse correlation between specific ALP activity and cell yield. Malonate-treated variations of specific ALP and of cell yield indices were up to 30- to 40-fold larger than variations within day 21 calcium precipitates. Thus, calcifying cells were almost unchanged relatively to noncalcifying cells. These results indicate that malonate-resistant cells are mostly selected, rather than induced, to differentiate by malonate. The results also show that stromal derived OPCs undergo a similar biochemical stage as in chondrocytes. © 1993 Wiley-Liss, Inc.     

Membrane junctions between salivary gland cells ofChironomus thummi

Summary Cells ofChironomus salivary glands communicate through intercellular connections of high permeability. Electron micrographs of salivary glands show two kinds of junctions between the membranes of adjacent cells, which may be responsible for cell coupling: septate junctions and close membrane junctions.A large fraction of lateral cell surfaces is occupied by septate junctions, while the area of close membrane junctions appears to be very small. Consequently septate junctions have been considered as likely sites for intercellular coupling. There are however some indications that intercellular communication is provided by structures which seem to be unstable. As osmotic effects are among the factors which can disrupt cellular communications, we have tried to eliminate possible effects of the fixing solutions on the ultrastructure of intercellular connections by using isoosmotic fixatives. Under these conditions large regions of close membrane junctions of the nexus kind have been observed to occur between gland cells. They are of similar size as septate junctions. It seems to be possible that as in other communicating cell systems nexus could be the sites for intercellular coupling of salivary gland cells.The authors would like to thank Prof. Dr. H. Leonhardt, Institut für Anatomie I, Homburg, for the use of his electron microscope (Zeiss EM 9-DFG grant LE 69–8) during part of this work and Prof. Dr. H. Kroeger, Institut für Genetik, Saarbrücken for the supply withChironomus larvae.     

Study of cell culture of mouse submandibular salivary gland in vitro

Cell culture keeping its phenotype till the 20th passage was obtained from mouse submandibular salivary glands. The analysis of the heterogeneous culture showed that there were some morphological types of cells: densely packed small cells that had cuboidal or polygonal form and also large round cells. Epithelial cells of submandibular gland cultured during several weeks were able to form tubular strucures. It was shown that glandulocyte culture was presented by K19-positive and NGF-positive cells. It is important to note that expression of genes coding proinsulin and insulin was detected by immunocytochemical staining and by PCR as well.     

Proteomic analysis of microvesicles derived from human colorectal cancer cells

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.     

Potential therapeutic effects of induced pluripotent stem cells on induced salivary gland cancer in experimental rats

Salivary gland neoplasms exhibit complex histopathology in a variety of tumor types and treatment options depend largely on the stage of the cancer. Induced pluripotent stem cells (iPS) have been investigated for treating induced salivary gland cancer and for restoring salivary gland function. We investigated iPS treatment for salivary gland cancer both in vitro and in vivo. For our study in vitro, we re-programmed human skin fibroblasts to form iPS cells using a plasmid containing Oct4, Sox2, L-MYC and LIN28. For our study in vivo, we used 30 white male albino rats divided into the following groups of 10: group 1 (control): rats were injected with phosphate-buffered saline (PBS), group 2 induced squamous cell carcinoma (SCC): rat submandibular glands were injected with squamous carcinoma cells (SCC), group 3 (induced SCC/iPS): SCC treated rats treated with 5 × 106 iPS cells. Submandibular glands from rats of all groups were examined histologically and real time PCR was performed for amylase, and COX I and COX II gene expression. We confirmed that submandibular gland specimens included tumor tissue before starting treatment with iPS. iPS treated cases exhibited regeneration of salivary glands, although minor degenerative and vascularization changes remained. The acinar cells regained their proper organization, but continued to exhibit abnormal activity including hyperchromatism. iPS cells may be useful for treating salivary gland carcinomas.