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carcinoma, transitional cell, immunotherapy, pembrolizumab, radiotherapy, treatment outcome, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, and RC254-282

Abstract Radiotherapy plus immune checkpoint inhibitors can potentially induce synergistic antitumor immune responses. However, little clinical evidence is established regarding their combination therapy. Here, we aimed to assess whether radiotherapy to the primary tumor impacts on the efficacy of pembrolizumab in advanced urothelial cancer. We retrospectively reviewed 98 advanced urothelial cancer patients receiving pembrolizumab in a second‐ or later‐line setting using our multicenter cohort. Patients were categorized according to a history of radiotherapy to the primary tumor: patients previously exposed to radiotherapy to the primary tumor (Radiotherapy group, 17 patients [17%]) and those not (Nonradiotherapy group, 81 patients [83%]). The associations of radiotherapy to the primary tumor with objective response and survival were evaluated. The Radiotherapy group showed a significantly higher objective response ratio than did the Non‐radiotherapy group (65% vs 19%; P

Bile duct, Morphogenesis, Liver metabolite, Bile recovery, Hierarchical co-culture, Biology (General), and QH301-705.5

Abstract Background Liver metabolites are used to diagnose disease and examine drugs in clinical pharmacokinetics. Therefore, development of an in vitro assay system that reproduces liver metabolite recovery would provide important benefits to pharmaceutical research. However, liver models have proven challenging to develop because of the lack of an appropriate bile duct structure for the accumulation and transport of metabolites from the liver parenchyma. Currently available bile duct models, such as the bile duct cyst-embedded extracellular matrix (ECM), lack any morphological resemblance to the tubular morphology of the living bile duct. Moreover, these systems cannot overcome metabolite recovery issues because they are established in isolated culture systems. Here, we successfully established a non-continuous tubular bile duct structure model in an open-culture system, which closely resembled an in vivo structure. This system was utilized to effectively collect liver metabolites separately from liver parenchymal cells. Results Triple-cell co-culture of primary rat hepatoblasts, rat biliary epithelial cells, and mouse embryonic fibroblasts was grown to mimic the morphogenesis of the bile duct during liver development. Overlaying the cells with ECM containing a Matrigel and collagen type I gel mixture promoted the development of a tubular bile duct structure. In this culture system, the expression of specific markers and signaling molecules related to biliary epithelial cell differentiation was highly upregulated during the ductal formation process. This bile duct structure also enabled the separate accumulation of metabolite analogs from liver parenchymal cells. Conclusions A morphogenesis-based culture system effectively establishes an advanced bile duct structure and improves the plasticity of liver models feasible for autologous in vitro metabolite-bile collection, which may enhance the performance of high-throughput liver models in cell-based assays.

Metal stent, Malignant ureteral obstruction, Ureteral stent, Metallic ureteric stenting, Resonance stent, Diseases of the genitourinary system. Urology, and RC870-923

Abstract Background To study the outcomes and experiences of using metallic stents in treating patients with malignant ureteral obstruction (MUO), we examined the effects of metallic ureteral stenting using the Cook Resonance® stent in the treatment of MUO. Methods All patients who had a Resonance metallic stent inserted between April 2015 and March 2018 at one of multiple facilities were prospectively observed with a 1-year follow-up. The primary outcome was the patency rate of the metallic ureteral stent. The secondary outcomes included the complications (e.g., infection and fever). Results Although stent insertion was attempted in 50 patients, the stent could not be inserted as a ureteral stent in three patients due to severe ureteral stricture, and one ureteral cancer patient was excluded from the analysis. The remaining 46 patients’ median age was 67 years (range 28–85 years) (16 males, 30 females). Twenty-four patients died during the study; their median survival time was 226 days. The median follow-up period for the censored patients was 355 days (range 16–372 days), and just seven patients were still alive without Resonance failure > 1 year later. The women’s IPSS scores tended to be lower than those of the men. Regarding the OABSS score, although the women’s total score tended to be low, the difference between the men’s and women’s scores was nonsignificant. The bacteria detected from urine culture after stent insertion were more gram-positive than gram-negative. Conclusion Metallic ureteric stenting using the Resonance stent is safe and effective for treating MUO. Subjective symptoms were relatively less in the female patients.

Medicine (General), R5-920, Cytology, and QH573-671

Suspension culture for the increase in human induced pluripotent stem cells (hiPSCs) has been one of the major challenges. Previously, we reported that albumin-associated lipids prevented aggregation of hiPSCs, whereas, lipids responsible for this function were unclear. Here, by using cell aggregation assay, we investigated principal lipids regulated aggregation size of hiPSCs. As a result, lysophosphatidic acid (LPA) and Sphingosine-1-phosphate (S1P), known as lysophospholipids acting as a signaling molecule, were identified. These lipids regulated the aggregation size in a dose-dependent manner. Aggregates formed with these lipids kept the high-expression rates of pluripotent marker genes and had the abilities of proliferation. These studies demonstrated that LPA and S1P were useful for suspension culture for hiPSCs without affecting the growth ability and pluripotency of hiPSCs. This knowledge will lead to the development of a simple and robust method for the mass culture of hiPSCs. Keywords: Suspension culture, Lysophospholipid, Aggregation, Pluripotent stem cells, Spheroid

Medicine (General), R5-920, Cytology, and QH573-671

A differentiation of human induced pluripotent stem cells (hiPSCs) into definitive endoderm linage is required for a preparation of metabolic organ derived cells. The differentiation consumed high-priced cytokines and small molecules, which have hampered the manufacturability of differentiated cells. Although the cytokines and small molecules are remained or cells produce the autocrine factors, daily culture medium change should be proceeded to remove toxic metabolites generated from cells. In this study, we developed a simple dialysis culture system to refine the medium during definitive endodermal differentiation. We demonstrated that dialysis culture prevented cell damage to remove lactate. The hiPSCs cultured with dialysis also differentiated similarly as usual differentiation without dialysis even if they were not supplied Activin A for latter culture days in the differentiation. With this dialysis culture system, hiPSCs were differentiated into endodermal lineage with medium refinement and recycling and autocrine factors as well as cytokines, which may lead to reduce differentiation cost. Keywords: Dialysis culture, Human induced pluripotent stem cell, Suspension culture, Definitive endoderm

Medicine (General), R5-920, Cytology, and QH573-671

Suspension culture of three-dimensional (3D) spheroid of human induced pluripotent stem cells (hiPSCs) has been known as a potential method to enhance the scalability of hepatic differentiation of hiPSCs. However, the impact of size-related factor of initial formed spheroid were not largely considered. To address this problem, we evaluate the impact of different specific spheroid size of hiPSCs by forming the individual spheroid from different number of hiPSCs and differentiated into hiPSCs-derived hepatocytes (iHeps). The results showed that larger spheroid exhibit enhanced capability to differentiated into hepatic lineage by increasing the expression marker albumin, CYP3A4 and lower expression of fetal hepatic marker AFP. Several factor such as the tendency of cystic like structure forming, the necrotic area of the large dense spheroid, and interference of WNT/β-catenin signaling was significantly affecting the resulted iHeps. In this study, we suggest that the optimal spheroid size for hepatic differentiation can be attained from 500 to 600 μm diameter spheroid formed from 12,500–25,000 hiPSCs. This size can be potentially applied for various practical use of hepatic differentiation in scalable suspension culture. Keywords: hiPSCs, iHeps, Spheroid, Size-dependent, Hepatic differentiation

hepatocyte, zonation, oxygen supply, metabolic reprogramming, ADME-Tox, PDMS, Biotechnology, and TP248.13-248.65

Pre-clinical drug screening is an important step in assessing the metabolic effects and hepatic toxicity of new pharmaceutical compounds. However, due to the complexity of the liver microarchitecture, simplified in vitro models do not adequately reflect in vivo situations. Especially spatial heterogeneity, known as metabolic zonation, is often lost due to limitations introduced by typical culture conditions. By culturing primary rat hepatocytes in varied ambient oxygen levels on either gas-permeable or non-permeable culture plates, we highlight the importance of biomimetic oxygen supply for the targeted induction of zonation-like phenotypes. Resulting cellular profiles illustrate the effect of pericellular oxygen concentration and consumption rates on hepatic functionality in terms of zone-specific metabolism and β-catenin signaling. We show that modulation of ambient oxygen tension can partially induce metabolic zonation in vitro when considering high supply rates, leading to in vivo-like drug metabolism. However, when oxygen supply is limited, similar modulation instead triggers an ischemic reprogramming, resembling metabolic profiles of hepatocellular carcinoma and increasing susceptibility toward drug-induced injury. Application of this knowledge will allow for the development of more accurate drug screening models to better identify adverse effects in hepatic drug metabolism.

5‐S‐cysteinyldopa, male, malignant melanoma, penis, urethra, Diseases of the genitourinary system. Urology, and RC870-923

Introduction We herein present a case of malignant melanoma of the male urethra with an increased serum 5‐S‐cysteinyldopa concentration. Case presentation A 77‐year‐old man visited our hospital complaining dysuria and a dark brown mass protruding from the external urethral meatus. His serum 5‐S‐cysteinyldopa concentration was elevated beyond the upper limit of the reference range. Biopsy of the tumor was performed, and the histological diagnosis was malignant melanoma. He underwent total penectomy, and the serum 5‐S‐cysteinyldopa concentration was normalized. He remained alive without evidence of locoregional recurrence or distant metastases for 6 months after surgery. Conclusion Malignant melanoma of the male urethra is uncommon. The prognosis is favorable if it is detected in its early stages. This case report suggests that measurement of the serum concentration of 5‐S‐cysteinyldopa, a melanin metabolite, is useful for early diagnosis of male urethral melanoma.

modular assembly, polyglycolic acid, selective laser sintering, tissue engineering, hepatoma Hep G2 cells., Technology, Engineering (General). Civil engineering (General), TA1-2040, Biology (General), QH301-705.5, Physics, QC1-999, Chemistry, and QD1-999

The aim of the present study was to design and fabricate polyglycolic acid (PGA) modules on the basis of the Raschig ring as a tissue element for bottom–top tissue engineering to increase the feasibility of cellular-assembly technology. Three types of modules, namely, cylindrical, Raschig ring, and transverse-pore modules, with different numbers and orientations of canals, were designed and fabricated by modified selective-laser-sintering (SLS) technology. These modules maintained their structure in a flowing culture environment, and degradation did not create an acidic environment, hence promoting their ability to scale up to highly functional tissue. The modules were seeded with human hepatoma Hep G2 cells and cultured for 10 days. The transverse-pore modules were found to have the highest glucose consumption, albumin production, and cell viability among the three tested modules. Our study showed that the proposed module design provided better mass transfer and possessed the required mechanical strength to enable use in the construction of large tissue.

Medicine and Science

Abstract Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 µm-size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-based assays.