Salivary Gland Tissue Recombination Can Modify Cell Fate.

Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate the roles of tissue interactions in cellular differentiation, we performed single-cell RNA sequencing and imaging analyses on recombined tissues, where mesenchyme and epithelium were switched ex vivo between two types of embryonic mouse salivary glands: the parotid gland (a serous gland) and the submandibular gland (a predominantly mucous gland). We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. The parotid epithelium recombined with submandibular mesenchyme began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be modified in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

Efficient Gene Knockout in Salivary Gland Epithelial Explant Cultures.

We have developed methods to achieve efficient CRISPR-Cas9-mediated gene knockout in ex vivo mouse embryonic salivary epithelial explants. Salivary epithelial explants provide a valuable model for characterizing cell signaling, differentiation, and epithelial morphogenesis, but research has been limited by a paucity of efficient gene perturbation methods. Here, we demonstrate highly efficient gene perturbation by transient transduction of guide RNA-expressing lentiviruses into Cas9-expressing salivary epithelial buds isolated from Cas9 transgenic mice. We first show that salivary epithelial explants can be cultured in low-concentration, nonsolidified Matrigel suspensions in 96-well plates, which greatly increases sample throughput compared to conventional cultures embedded in solidified Matrigel. We further show that salivary epithelial explants can grow and branch with FGF7 alone, while supplementing with insulin, transferrin, and selenium (ITS) enhances growth and branching. We then describe an efficient workflow to produce experiment-ready, high-titer lentiviruses within 1 wk after molecular cloning. To track transduced cells, we designed the lentiviral vector to coexpress a nuclear fluorescent reporter with the guide RNA. We routinely achieved 80% transduction efficiency when antibiotic selection was used. Importantly, we detected robust loss of targeted protein products when testing 9 guide RNAs for 3 different genes. Moreover, targeting the ß1 integrin gene (Itgb1) inhibited branching morphogenesis, which supports the importance of cell-matrix adhesion in driving branching morphogenesis. In summary, we have established a lentivirus-based method that can efficiently perturb genes of interest in salivary epithelial explants, which will greatly facilitate studies of specific gene functions using this system.

Single-Cell RNA-seq Identifies Cell Diversity in Embryonic Salivary Glands.

Branching organs, including the salivary and mammary glands, lung, and kidney, arise as epithelial buds that are morphologically very similar. However, the mesenchyme is known to guide epithelial morphogenesis and to help govern cell fate and eventual organ specificity. We performed single-cell transcriptome analyses of 14,441 cells from embryonic day 12 submandibular and parotid salivary glands to characterize their molecular identities during bud initiation. The mesenchymal cells were considerably more heterogeneous by clustering analysis than the epithelial cells. Nonetheless, distinct clusters were evident among even the epithelial cells, where unique molecular markers separated presumptive bud and duct cells. Mesenchymal cells formed separate, well-defined clusters specific to each gland. Neuronal and muscle cells of the 2 glands in particular showed different markers and localization patterns. Several gland-specific genes were characteristic of different rhombomeres. A muscle cluster was prominent in the parotid, which was not myoepithelial or vascular smooth muscle. Instead, the muscle cluster expressed genes that mediate skeletal muscle differentiation and function. Striated muscle was indeed found later in development surrounding the parotid gland. Distinct spatial localization patterns of neuronal and muscle cells in embryonic stages appear to foreshadow later differences in adult organ function. These findings demonstrate that the establishment of transcriptional identities emerges early in development, primarily in the mesenchyme of developing salivary glands. We present the first comprehensive description of molecular signatures that define specific cellular landmarks for the bud initiation stage, when the neural crest-derived ectomesenchyme predominates in the salivary mesenchyme that immediately surrounds the budding epithelium. We also provide the first transcriptome data for the largely understudied embryonic parotid gland as compared with the submandibular gland, focusing on the mesenchymal cell populations.

Phase I study of LY2181308, an antisense oligonucleotide against survivin, in patients with advanced solid tumors.

PURPOSE: LY2181308 is an antisense oligonucleotide that complementarily binds to survivin mRNA and inhibits its expression in tumor tissue. This phase I dose escalation study evaluated the tolerability, pharmacokinetics, and anticancer activity of LY2181308 in Japanese. METHODS: Patients with solid tumors refractory to standard therapy received LY2181308 (400, 600, or 750 mg) as a 3-h intravenous infusion for 3 consecutive days and thereafter once a week. RESULTS: LY2181308 was administered to 14 patients, aged 44-73 (median 60) years. Flu-like syndrome, prolonged prothrombin time-international normalized ratio (PT-INR), thrombocytopenia, and fatigue were common reversible grade 1/2 toxicities. The dose-limiting toxicity was reversible grade 3 elevation of ALT/AST/γ-GTP in 1 patient treated at the 750-mg dose. Pharmacokinetic analysis showed a long terminal half-life of 21 days and an extensive tissue distribution of LY2181308. In 12 evaluable patients, one patient had stable disease, while the remaining 11 patients had progressive disease. CONCLUSIONS: LY2181308 monotherapy is well tolerated up to 750 mg with a manageable toxicity, the pharmacokinetic profile warrants further evaluation of LY2181308 in combination with cytotoxic agents or radiotherapy.

Thoracic cage extent of melorheostosis depicted by multislice CT.

The ribs and vertebrae are rarely affected by melorheostosis. We present a case of melorheostosis of the ribs and thoracic vertebrae, in which multislice computed tomography (CT) was useful in depicting its extent and defining its sclerotome distribution.

[Multislice CT of the thorax: effects of contrast material pushed with saline solution using a dual power injector on contrast material dose and perivenous artifacts].

A new method of power injection of contrast material flushed with saline solution for thoracic multislice CT using a dual power injector was evaluated in 105 patients. The patients were categorized into 3 groups of 35 patients each, according to the protocol of contrast material administration: (1) 100 mL of non-ionic contrast material(300 mgI/mL), (2) 75 mL of the same contrast material, and (3) 75 mL of the same contrast material flushed with 30 mL of saline solution. Scanning was performed in a caudal-to-cranial direction. Mean attenuation for the three protocols was measured in the superior vena cava, pulmonary trunk, ascending aorta, and descending aorta. Vascular opacification and perivenous artifacts were graded using four-point scoring by a consensus panel of three blinded radiologists. Intravenous injection of 75 mL of contrast material flushed with saline solution provided significantly better vascular opacification in the superior vena cava(p < 0.001) and pulmonary trunk(p < 0.02) than that provided by a 75 mL or 100 mL injection of contrast material alone. A similar degree of enhancement was observed in the ascending and descending aorta. Further, perivenous artifacts in the subclavian vein were significantly reduced (p < 0.05).

Japanese experience of telemedicine in oncology.

We started telemedicine projects from 1990 with a telepathology system within Tsukiji Campus of National Cancer Center. In 1994, we connected Tsukiji Campus and Kashiwa Campus by 6 Mbps optical fiber leased line using IP protocol for data transmission, for teleconference, telepathology, and teleradiology projects. We also started connection of regional cancer centers and are now forming a cancer center network of 14 cancer centers. We are at present organizing 130 teleconferences per year with an attendance of more than 16000 people as summary. We have also used a high-resolution image transferring system, such as SHD (2000 pixelsx2000 pixels resolution) system on one side, and an economical telemedicine system using JAVA and a WWW browser (NCC_image) on the other side. We think that providing information is another field of telemedicine. We began the experimental gopher and WWW service in 1993. We are now providing official up-to-date cancer information for patients and healthcare professionals. We are getting more than 400000 hits per month. We are also providing a teleconference video session which is held every week on the Internet using a Real Video system with synchronized slide presentation on the WWW browser. We are also organizing a Cancer Image Reference Database System including DICOM images with viewer software. This paper is a summary of the telemedicine projects performed at the National Cancer Center.

Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry.

Quantitative determination by liquid chromatography (LC) coupled with mass spectrometry (MS) was achieved for the following 10 toxins found in association with diarrhetic shellfish poisoning: okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoylokadaic acid (palOA), 7-O-palmitoyldinophysistoxin-1 (pa1DTX1), pectenotoxin-1 (PTX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2SA), pectenotoxin-6 (PTX6), yessotoxin (YTX), and 45-hydroxyyessotoxin (YTXOH). Toxins in 2 g of the adductor muscle or the digestive glands of scallops, Patinopecten yessoensis, were extracted with 18 ml of methanol-water (9:1, v/v), freed of polar contaminants by partition between chloroform and water, and treated by solid-phase extraction on a silica cartridge column. Samples containing YTXOH were purified separately on a buffered reversed-phase column. Chromatographic separation was achieved by the following combinations of columns and mobile phases: a Symmetry C18 column with acetonitrile-0.05% acetic acid (7:3, v/v) for OA, DTX1, PTX6 and PTX2SA; a Develosil ODS column with the same mobile phase for PTX1 and PTX2; a Capcellpak column with methanol-2.5% acetic acid (98:2, v/v) for palOA and palDTX1; and an Inertsil ODS column with methanol-0.2 M ammonium acetate (8:2, v/v) for YTX and YTXOH. Carboxylic acid toxins were selectively monitored on [M-H]- ions, sulfated toxins on [M-Na]-ions, and neutral toxins on [M+NH4]+ ions. Average recoveries of the toxins spiked to tissue homogenates ranged from 70 to 134%. Detection limits in the muscle ranged from 5 to 40 ng/g and those in the digestive glands from 10 to 80 ng/g.

Intraoperative and conformal external-beam radiation therapy in patients with locally advanced pancreatic carcinoma; results from a feasibility phase II study.

BACKGROUND/AIMS: Chemoradiation therapy is widely indicated to patients with locally advanced pancreatic carcinoma, though the capability of radiotherapy alone is not assessed enough. The purpose of this study is to clarify the efficacy and safety of a more intensive radiotherapy for those patients. METHODOLOGY: Fifteen patients were enrolled in a feasible phase II study of treatment with intraoperative radiation therapy (25 Gy), followed by conformal external-beam radiation therapy (40 Gy in 20 fractions, 5 times/week). The antitumor effect was evaluated on the early phase of dynamic computed tomography image. RESULTS: The full irradiation dose was feasible in 12 of 15 patients. The overall response rate was 40% (1 complete and 5 partial responses). Grade 3 toxicity was observed in 2 patients (13%) with nausea/vomiting or anorexia. One patient developed gastric ulcer and died of gastrointestinal bleeding 12 months after intraoperative radiation therapy. The median survival time was 11.1 months, and the 2-year survival rates were 13%. Survival for more than 2 years was observed in 2 of the 6 responders. CONCLUSIONS: The above radiotherapy is considered to be active for the locally advanced pancreatic cancer with acceptable toxicity, when the gastrointestinal tract is excluded from the radiation field. This should be further assessed in late phase II studies involving a large number of patients.

Assessment of arterial tumor vascularity in small hepatocellular carcinoma. Comparison between color doppler ultrasonography and radiographic imagings with contrast medium: dynamic CT, angiography, and CT hepatic arteriography.

Hepatocellular carcinoma (HCC) is characterized by tumor vascularization from the hepatic artery. The objective of our work was to compare color Doppler ultrasonography (CDU), including power Doppler ultrasonography (PDU) with radiographic imagings with contrast medium in regard to the detection of the arterial tumor vascularity of small hepatocellular carcinomas (HCC). We examined 42 small HCC lesions 2 cm or less in diameter in 37 patients for arterial tumor vascularity by conventional CDU, PDU, dynamic computed tomography (dCT), digital subtraction angiography (DSA), and CT hepatic arteriography (CTA). Color images were detected in 25 (59.5%) and 28 (66.7%) of the 42 lesions with conventional CDU and PDU, respectively, and tumor vascularity was detected in 26 (61.9%) by dCT, 23 (54.8%) by DSA, and 29 (69.0%) by CTA. Tumor vascularity could be detected in 51.9% by PDU and CTA, more than by conventional CDU, dCT, and DSA (44.4, 44.4, and 33.3%, respectively) in well-differentiated HCC, whereas the detection rates by these techniques were almost equal (86.7% by CDU, 93.3% by PDU, dCT, and DSA, 100% by CTA, respectively) in moderately and poorly differentiated HCC. PDU is superior to CDU, dCT and DSA and equal to CTA for the detection of tumor vascularity in small HCC, particularly in well-differentiated HCC.

Diagnosis of gastric cancers: comparison of conventional radiography and digital radiography with a 4 million-pixel charge-coupled device.

PURPOSE: To evaluate the differences in accuracy and observer performance at conventional radiography and at digital radiography with a 4 million-pixel charge-coupled device (CCD) for the diagnosis of gastric cancers. MATERIALS AND METHODS: A prospective study was performed of 225 patients with suspected gastric cancer who were referred to our hospital from January 1997 through February 1997. One hundred twelve patients were examined at conventional radiography and 113 were examined at digital radiography, and 24 and 27 patients had gastric cancer, respectively. Six radiologists interpreted the images, with attention to tumor findings. They were blinded to the clinical details, and their interpretations were rated against those of three other radiologists who examined the patients and who were aware of the clinical information such as endoscopic features and/or histopathologic findings in biopsy specimens. Receiver operating characteristic (ROC) analysis was used to compare the differences in observer performance for the diagnosis of gastric cancers at conventional radiography and at digital radiography. RESULTS: The overall sensitivity was 64.6% at conventional radiography versus 75.3% at digital radiography (P =. 287); specificities were 84.5% and 90.5%, respectively (P =.011); and the positive predictive values were 53.1% and 71.3%, respectively (P =.036). ROC analysis clearly showed higher diagnostic performance at digital radiography than at conventional radiography. CONCLUSION: The data demonstrate the high diagnostic value of digital radiography with a 4 million-pixel CCD for gastric cancers. The technique has considerable potential as an alternative to conventional gastrointestinal radiography.

Optimal injection protocol for CT evaluation during hepatic arterial infusion chemotherapy.

OBJECTIVE: As an imaging modality for follow-up during continuous or repeated hepatic arterial infusion chemotherapy using a hepatic intra-arterial indwelling catheter, the usefulness of CT while infusing contrast through the indwelling catheter (reservoir port) was examined. METHODS: Using reservoir ports implanted in eight patients with hepatic metastasis from colon cancer, radioisotope perfusion scintigraphy (RI), CT (three rates of infusion of contrast were used), and digital subtraction angiography (AG) were performed to compare the modalities' ability to visualize the intrahepatic and abnormal extrahepatic distributions. RESULTS: CT (infusion rate 0.1 mL/sec) was superior to AG and RI in terms of the ability to visualize intrahepatic distribution, particularly in small areas, and facilitated 3D delineation of the distribution. In evaluating extrahepatic distribution, CT also outperformed the other modalities. CONCLUSIONS: For imaging study follow-up during hepatic arterial infusion chemotherapy, CT proved to be more useful than conventional RI and AG.

[Staging of pancreatic ductal adenocarcinoma using dynamic MR imaging].

Single breath-hold gradient echo images were obtained before and immediately after bolus intravenous administration of Gd-DTPA (dynamic MR imaging) in the study of the pancreas. Of 37 patients with pathologically proved pancreatic ductal adenocarcinoma, seventeen patients who underwent both dynamic MR imaging studies and curative surgery were included in this study. Correlations between histologic findings in the resected specimens and MR images were analyzed as to tumor extension and staging according to the General Rules for the Study of Pancreatic Cancer (4th Edition) published by the Japan Pancreas Society. In comparison with conventional MR images, dynamic MR imaging improved the detectability of pancreatic carcinoma and delineation of the vasculature by clarifying the margin of the tumor and the vessels. Nonenhanced T1-weighted imaging is the best sequence to estimate peripancreatic tumor extension, because the contrast between the tumor and peripancreatic fat deteriorates with the use of contrast material. There is a tendency to overestimate vascular invasion on MR images, the reason for which is considered to be the contractive nature of fibrotic change induced by pancreatic carcinoma. The diagnostic efficacy of lymph node metastasis remains insufficient on MR images because some cases show no enlargement of lymph nodes in spite of the existence of pathological metastasis. Our results suggest that dynamic MR imaging has the advantage of improving the conspicuity of the tumor and the vasculature.

[Effects of NMDA antagonist, MK-801 on spatial exploratory behavior in the rat].

Rats given an antagonist against N-methyl-D-aspartate, (+)-10, 11-dihydro-5-methyl-5H-debenzo (a, d) cycloheptene-5, 10-imine (MK-801), were compared with control rats for their activity and exploratory behavior (habituation, exploration time to the spatial change of one of 4 objects and to the new object) in a circular open field. Rats given 0.07 mg/kg dose of MK-801 displayed no significant differences with the controls. Rats given 0.1 mg/kg dose of MK-801 failed to respond to the spatial change, whereas they displayed habituation and exploration to the new object at the same degree as the control rats. Rats given 0.3 mg/kg dose of MK-801 displayed hyperactivity and did not display habituation and exploration. The result suggests that the 0.1 mg/kg dose of MK-801, which dose not affect on activity, habituation and exploration to the new object in rats, selectively affects on acquisition of spatial information and reduces their spatial exploration.

Intracranial metastases of hepatocellular carcinoma: CT and MRI.

We report clinical characteristics and CT and MRI in 16 patients with brain metastases due to hepatocellular carcinoma (HCC). Eight of these 16 patients presented with apoplexy-like symptoms (50%). Pulmonary metastases were found in 13 cases (81.3%). The mean survival from the appearance of cerebral metastases to death was 6.2 weeks, which is one of the shortest survival terms in metastatic brain tumours. Haemorrhagic brain metastases were observed in 14 patients (87.5%) with a tendency for the frequency of bleeding to increase in proportion to the size of the tumour. On both contrast-enhanced CT and MRI, metastatic brain tumours enhanced strongly, suggesting that brain metastases, like HCC, are also hypervascular. MRI is useful in evaluating brain metastases from HCC, especially in order to differentiate tumour from haemorrhage. Our results demonstrated a poor prognosis and bleeding tendency of brain metastases due to HCC and showed the usefulness of CT and MRI in achieving a correct diagnosis.

Virtual reality enhanced surgical conference system.

We present our ongoing work on an enhanced surgical conference system with a technology of virtual reality (VR). We reported on a surgical simulation support system by using a technology of virtual reality last year. In the present time, while using our VR simulation system, we realized that many surgeons and nurses needed to see both a solid real image and a virtual image of the surgical operation at the same time. According to this reason we added a solid video system to our previous VR simulation system. The new system can display both real and virtual images on 100 inch wide screen and a console monitor of Onyx computer. The doctors can see both images with shutter glasses on the screen or console. We can now simulate various cancer surgery while watching the real solid surgical picture. We expect our enhanced surgical conference system to be beneficial for surgeons and nurses with limited experience to familiarize surgical procedures. The system could be also employed in planning a surgical procedure and educating medical staffs. Here we discuss about the aim of the system, current implementation, its limitations and its future directions.