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Supplementary Figure 3 from Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

PDF file - 142K, Contains Luciferase assay, Western blotting, ELISA, Immunoblotting, YY2 gene silencing, Cell viability, Quantitative PCR, and Immunoprecipitation data.

Supplementary Figure 1 from Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

Supplementary Figure 2 from Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

PDF file - 270K, The proliferation property of HeLa cells, wild type HCT116 (+/+) cells, and p53-null HCT116 (-/-) cells under hypoxic condition

PDF file - 60K, Primer pairs used in this study for quantitative Real-time RT-PCR

Supplementary Figure Legends 1-3 from Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

PDF file - 101K, YY1-silencing suppresses the colonization and growth of A549-luc cells in lungs

Supplementary Table 1 from Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis. [Cancer Res 2007;67(21):10181–9]

Supplementary Figures 1-12 from Autophagy Is Activated by TGF-β and Potentiates TGF-β–Mediated Growth Inhibition in Human Hepatocellular Carcinoma Cells

PDF file - 129K, The establishment of HCT116 p53-/-/shYY1 stable cells

PDF file - 57K, YY1 overexpression does not affect the expression of HIF-1α under hypoxia

Supplementary Figure Legends 1-12 from Autophagy Is Activated by TGF-β and Potentiates TGF-β–Mediated Growth Inhibition in Human Hepatocellular Carcinoma Cells

PDF file - 127K, shYY1 vectors significantly silence YY1 expression

PDF file - 173K, The effect of YY2-silencing on HIF-1a target genes and the effect of YY1-silencing on hydroxyl HIF-1a under hypoxic condition

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates cell growth, differentiation, and apoptosis of various types of cells. Autophagy is emerging as a critical response of normal and cancer cells to environmental changes, but the relationship between TGF-β signaling and autophagy has been poorly understood. Here, we showed that TGF-β activates autophagy in human hepatocellular carcinoma cell lines. TGF-β induced accumulation of autophagosomes and conversion of microtubule-associated protein 1 light chain 3 and enhanced the degradation rate of long-lived proteins. TGF-β increased the mRNA expression levels of BECLIN1, ATG5, ATG7, and death-associated protein kinase (DAPK). Knockdown of Smad2/3, Smad4, or DAPK, or inhibition of c-Jun NH2-terminal kinase, attenuated TGF-β–induced autophagy, indicating the involvement of both Smad and non-Smad pathway(s). TGF-β activated autophagy earlier than execution of apoptosis (6-12 versus 48 h), and reduction of autophagy genes by small interfering RNA attenuated TGF-β–mediated growth inhibition and induction of proapoptotic genes Bim and Bmf, suggesting the contribution of autophagy pathway to the growth-inhibitory effect of TGF-β. Additionally, TGF-β also induced autophagy in some mammary carcinoma cells, including MDA-MB-231 cells. These findings show that TGF-β signaling pathway activates autophagy in certain human cancer cells and that induction of autophagy is a novel aspect of biological functions of TGF-β. [Cancer Res 2009;69(23):8844–52]

Supplementary Tables 1-2 from Autophagy Is Activated by TGF-β and Potentiates TGF-β–Mediated Growth Inhibition in Human Hepatocellular Carcinoma Cells

PDF file - 91K

Background The incidence of nontuberculous mycobacterial (NTM) infections has been increasing worldwide, becoming a significant healthcare burden especially among elderly people. This study aimed to evaluate the trends in NTM-associated mortality in Japan. Methods This study used vital statistics data and data on all NTM-associated deaths (N = 18 814) among individuals aged ≥40 years in Japan from 1997 to 2016. We calculated the crude and age-adjusted mortality rates by age and sex and used joinpoint regression to analyze trends and estimate the average annual percentage change (AAPC). We compared crude NTM- and tuberculosis-associated mortality rates by sex. Results The overall crude annual mortality rate increased from 0.63/100 000/year in 1997 to 1.93/100 000/year in 2016 and was the highest among individuals aged 80–84 years. The AAPC of the crude mortality rates among men of all ages and women aged 40–59 years were stable but increased among women aged 60–79 years (3.5%; 95% confidence interval [CI], 2.8–4.3) and ≥80 years (4.3%; 95% CI, 3.7–4.9). Among men, the age-adjusted mortality rates did not show a significant trend, while among women, the rates increased over the study period (AAPC, 4.6%; 95% CI, 2.7–6.6). In women, the crude NTM-associated mortality rate exceeded the tuberculosis mortality rate in 2014, 2015, and 2016. Conclusions NTM mortality increased in Japan between 1997 and 2016, especially among the elderly female population. Given the increasing NTM-associated mortality and the susceptible aging population, public health authorities in Japan should pay greater attention to NTM infections.

BACKGROUND AND AIM The current prevalence of hepatitis C virus infection and hepatitis C virus-associated mortality in Japan falls short of the World Health Organization goal of viral hepatitis elimination by 2030. We aimed to evaluate the trends in hepatitis C virus-associated mortality in Japan. METHODS This nationwide observational study used the Japanese Vital Statistics from 1998 to 2017 and included all Japanese hepatitis C virus-associated deaths (84 936) of adults aged ≥ 40 years. We calculated the crude and age-standardized mortality rates per 100 000 persons by age and sex. Joinpoint regression analysis was used to identify significant changing points in trends and to estimate the annual percentage changes and the average annual percentage changes for the entire study period. RESULTS The crude mortality rate per 100 000 persons (annual death number) increased from 5.5 (3548) in 1998 to 7.0 (4843) in 2005 and decreased to 4.0 (3095) in 2017. By 2017, the crude mortality rates per 100 000 persons among men and women had dropped to 3.6 and 4.3, respectively. The age-standardized mortality rate was higher in women than in men. The average annual percentage change was -3.8% (95% confidence interval: -5.0 to -2.5). The declining trend was more rapid in men (-4.5%, 95% confidence interval: -5.3 to -3.6) than in women (-2.7%, 95% confidence interval: -3.8 to -1.6). CONCLUSIONS Trends in hepatitis C virus-associated mortality rates have declined in an accelerating manner in Japan, especially among men.

Adverse drug events (ADEs) are a major cause of mortality. We examined long-term trends for ADE-related deaths in Japan. This observational study was conducted using the Japanese Vital Statistics from 1999 to 2016. Data for all ADE-related deaths were extracted using International Classification of Diseases, Tenth Revision codes. We analysed ADE-related deaths by age and sex and calculated crude and age-standardised mortality rates (ASMR) per 100,000 people. We used Joinpoint regression analysis to identify significant changing points in mortality trends and to estimate annual percentage change (APC). In total, 16,417 ADE-related deaths were identified. The crude mortality rate for individuals aged ≥ 65 years was higher than that of young individuals. The ASMR per 100,000 people increased from 0.44 in 1999 to 0.64 in 2016. The crude mortality rate increased from 0.44 in 1999 to 1.01 in 2016. The APC of ASMR increased at a rate of 2.8% (95% confidence interval [CI] 1.4–4.2) throughout the study period. In addition, crude mortality increased at a rate of 5.7% (95% CI 4.2–7.3) annually from 1999 to 2016. The ADE-related mortality rate was higher for men than for women during the study period. The number of and trend in ADE-related deaths increased in Japan from 1999 to 2016, particularly in the older population. Adverse drug events (ADEs) are a public health issue, but descriptive data on ADEs in Japan are limited. Studies have shown that elderly people have a higher risk of dying from ADEs. Japan has one of the most rapidly aging populations and the highest percentage of older individuals worldwide. Clarifying long-term data trends in Japan is important in the aging world. Here, we aimed to clarify the trend in mortality related to ADEs in Japan. We selected 16,417 deaths that were assigned an underlying cause (i.e., ADEs) in vital statistics based on codes from the International Classification of Diseases, Tenth Revision (ICD-10). The crude mortality rate for both sexes increased from 0.44 per 100,000 in 1999 to 1.01 in 2016. The average annual percentage change (average APC), which numerically shows the change over time, was 5.7% throughout the study period. The age-standardised mortality rate, using the population in the first year, increased from 0.44 per 100,000 in 1999 to 0.64 in 2016. The average APC of the age-standardised mortality rate showed an increasing trend at 2.8%. Even after age standardisation, ADE-associated death showed an increasing trend. In particular, population groups aged ≥65 years showed a continuous increasing trend. These findings suggest that the ADE-related mortality rate in Japan is increasing, especially in elderly individuals.

Appropriate antibiotic prescriptions for outpatients with acute respiratory tract infections (ARTIs) are urgently needed in Japan. However, the empirical proof of this need is under-documented. Therefore, we aimed to determine antibiotic prescription rates, and the proportions of antibiotic classes prescribed for Japanese patients with ARTIs.We analysed health insurance claims data over 2013-2015 among Japanese patients aged75 years and determined the following indicators: 1) visit rates for patients with ARTIs and antibiotic prescription rates per 1000 person-years, and 2) proportion of visits by antibiotic-prescribed patients with ARTIs. We defined broad-spectrum antibiotics using the WHO Anatomical Therapeutic Chemical classification 4 level codes.Among 8.65 million visits due to ARTIs at 6859 hospitals and 62,024 physicians' offices, the visit rate and antibiotic prescription rate per 1000 person-years were 990.6 (99% confidence interval [CI], 989.4-991.7) and 532.4 (99% CI, 531.6-533.3), respectively. The visit rates for patients aged 0-17, 18-59, and 60-74 years were 2410.0 (99% CI, 2407.2-2412.9), 683.6 (99% CI, 682.7-684.6), and 682.1 (99% CI, 678.2-686.0), and antibiotic prescription rates were 1093.3 (99% CI, 1091.4-1095.2), 434.1 (99% CI, 433.4-434.9), and 353.4 (99% CI, 350.7-356.1), respectively. The overall proportion of antibiotic prescriptions for ARTI visits was 52.7% and 91.3% of the antibiotics prescribed were broad-spectrum.Both the visit rates and antibiotic prescription rates for ARTIs were high in this Japanese cohort. The proportion of antibiotic prescriptions exceeded that recommended in the clinical guidelines. Thus, there might be a scope for reducing the current antibiotic prescription rate in Japan.

Simple Summary Pancreatic cancer is difficult to treat. Novel treatment strategies are urgently needed to improve the survival rate, which is approximately 10% five years after diagnosis. The use of nanomedicines, which are formulated within a characteristic size range that favors its specific delivery to the diseased tissue, is being actively explored in cancer treatment. However, fibrosis (the abnormal accumulation of a cell type called fibroblasts and the fibrous protein network that they create) is characteristically seen in pancreatic cancer and hinders the delivery of nanomedicines into cancerous tissue. The decreased efficiency of delivery limits the therapeutic effects of nanomedicine in pancreatic cancer. We call this the "fibrotic barrier" to nanomedicine. To overcome the fibrotic barrier, we could target the fibrotic process and/or optimize the nanomedicine design. In this review, we give a detailed overview of strategies to overcome the fibrotic barriers in pancreatic cancer and highlight key gaps in our understanding. Pancreatic cancer is notorious for its dismal prognosis. The enhanced permeability and retention (EPR) effect theory posits that nanomedicines (therapeutics in the size range of approximately 10-200 nm) selectively accumulate in tumors. Nanomedicine has thus been suggested to be the "magic bullet"-both effective and safe-to treat pancreatic cancer. However, the densely fibrotic tumor microenvironment of pancreatic cancer impedes nanomedicine delivery. The EPR effect is thus insufficient to achieve a significant therapeutic effect. Intratumoral fibrosis is chiefly driven by aberrantly activated fibroblasts and the extracellular matrix (ECM) components secreted. Fibroblast and ECM abnormalities offer various potential targets for therapeutic intervention. In this review, we detail the diverse strategies being tested to overcome the fibrotic barriers to nanomedicine in pancreatic cancer. Strategies that target the fibrotic tissue/process are discussed first, which are followed by strategies to optimize nanomedicine design. We provide an overview of how a deeper understanding, increasingly at single-cell resolution, of fibroblast biology is revealing the complex role of the fibrotic stroma in pancreatic cancer pathogenesis and consider the therapeutic implications. Finally, we discuss critical gaps in our understanding and how we might better formulate strategies to successfully overcome the fibrotic barriers in pancreatic cancer.

Background Oral anticoagulants use has increased rapidly, internationally. Here we look at risks and benefits, based on Japanese data, of therapy with low risk non-valvular atrial fibrillation patients. Objectives Using a health insurance claims data set we assessed: (i) oral anticoagulants usage in Japan, and (ii) efficacy and safety of dabigatran compared with warfarin, in Japanese patients with non-valvular atrial fibrillation, aged 18–74 years. Methods We identified 4380 non-valvular atrial fibrillation patients treated with anticoagulants between 1 January 2005, and 28 February 2014, and estimated the adjusted hazard ratio for stroke or systemic embolism, and any hemorrhagic event (Cox proportional hazards regression model with stabilized inverse probability treatment weighting). Results The data included 101 989 anticoagulant prescriptions for 4380 patients, of which direct oral anticoagulants increased to 40.0% of the total by the end of the study. After applying exclusion criteria, 1536 new non-valvular atrial fibrillation patients were identified, including 1071 treated with warfarin and 465 with dabigatran. Mean ages were 56.11 ± 9.70 years for warfarin, and 55.80 ± 9.65 years for dabigatran. The adjusted hazard ratio (95% confidence interval), comparing dabigatran with warfarin, was 0.48 (0.25–0.91) for stroke or systemic embolism, and 0.91 (0.60–1.39) for any hemorrhage including intracranial and gastrointestinal. Conclusions Number of patients prescribed direct oral anticoagulants steadily increased, and incidence of all-cause bleeding related to dabigatran was similar to warfarin, in our study population of younger non-valvular atrial fibrillation patients. Dabigatran, compared with warfarin, generally reduced risk of all-cause stroke and systemic embolism.

Desmoplasia is a hallmark of pancreatic cancer and consists of fibrotic cells and secreted extracellular matrix (ECM) components. Various in vitro three-dimensional (3D) models of desmoplasia have been reported, but little is known about the relevant thickness of the engineered fibrotic tissue. We thus measured the thickness of fibrotic tissue in human pancreatic cancer, as defined by the distance from the blood vessel wall to tumor cells. We then generated a 3D fibrosis model with a thickness reaching the clinically observed range using pancreatic stellate cells (PSCs), the main cellular constituent of pancreatic cancer desmoplasia. Using this model, we found that Collagen fiber deposition was increased and Fibronectin fibril orientation drastically remodeled by PSCs, but not normal fibroblasts, in a manner dependent on Transforming Growth Factor (TGF)-β/Rho-Associated Kinase (ROCK) signaling and Matrix Metalloproteinase (MMP) activity. Finally, by targeting Secreted Protein, Acidic and Rich in Cysteine (SPARC) by siRNA, we found that SPARC expression in PSCs was necessary for ECM remodeling. Taken together, we developed a 3D fibrosis model of pancreatic cancer with a clinically relevant thickness and observed aberrant SPARC-dependent ECM remodeling in cancer-derived PSCs.

Background: Globally, the number of deaths is estimated to increase to 74 million per year by 2030. Place of death (PoD) is increasingly being recognized as an important aspect of end-of-life care....

In pulmonary arterial hypertension (PAH), excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) causes vascular medial thickening. Medial thickening is a histopathological hallmark of pulmonary vascular remodeling, the central disease process driving PAH progression. Pulmonary vascular remodeling causes stenosis and/or obstruction of small pulmonary arteries. This leads to increased pulmonary vascular resistance, elevated pulmonary arterial pressure, and ultimately right heart failure. To improve the survival of PAH patients, which remains at approximately 60% at 3 years after diagnosis, the development of novel PAH-targeted drugs is desired. To this end, a detailed understanding of the mechanisms underlying excessive PASMC proliferation and the medial thickening that ensues is necessary. However, a lack of in vitro models that recapitulate medial thickening impedes our deeper understanding of the pathogenetic mechanisms involved. In the present study, we applied 3-dimensional (3D) cell culture technology to develop a novel in vitro model of the pulmonary artery medial layer using human PAH patient-derived PASMCs. The addition of platelet-derived growth factor (PDGF)-BB, a mitogen known to promote excessive PASMC proliferation in PAH, resulted in increased thickness of the 3D-PAH media tissues. Conversely, administration of the PDGF receptor inhibitor imatinib or other clinical PAH drugs inhibited this medial thickening-inducing effect of PDGF-BB. Altogether, by using 3D cell culture technology, we report the generation of an in vitro model of medial thickening in PAH, which had hitherto not been successfully modeled in vitro. This model is potentially useful for assessing the ability of candidate PAH drugs to suppress medial thickening.

Pancreatic ductal adenocarcinoma (PDAC) is an often lethal disease characterized by a dense, fibrotic stroma. However, the lack of relevant preclinical models that recapitulate the characteristic histopathology of human PDAC in vitro impedes the development of novel therapies. The amount of stromal elements differ largely within and between patients, but in vitro models of human PDAC often do not account for this heterogeneity. Indeed, analyses of human PDAC histopathology revealed that the proportion of stroma ranged from 40 to 80% across patients. We, therefore, generated a novel 3D model of human PDAC, consisting of co-cultured human PDAC tumor cells and fibroblasts/pancreatic stellate cells, in which the proportion of fibrotic elements can be tuned across the clinically observed range. Using this model, we analyzed the signaling pathways involved in the differentiation of myofibroblasts, a characteristic subpopulation of fibroblasts seen in PDAC. We show that both YAP and SMAD2/3 in fibroblasts are required for myofibroblastic differentiation and that both shared and distinct signaling pathways regulate the nuclear localization of these factors during this process. Our novel model will be useful in promoting the understanding of the complex mechanisms by which the fibrotic stroma develops and how it might be therapeutically targeted.

Tumour models mimicking in vivo three-dimensional (3D) microenvironments are of increasing interest in drug discovery because of the limitations inherent to current models. For example, preclinical assays that rely on monolayer or spheroid cell cultures cannot easily predict 3D cancer behaviours because they have no vasculature. Furthermore, there are major differences in cancer behaviour between human and animal experiments. Here, we show the construction of 3D blood/lymph-vascularized human stromal tissues that can be combined with cancer cells to mimic dynamic metastasis for real-time throughput screening of secreted proteinases. We validated this tool using three human carcinoma cell types that are known to invade blood/lymph vessels and promote angiogenesis. These cell types exhibited characteristic haematogenous/lymphogenous metastasis and tumour angiogenesis properties. Importantly, these carcinoma cells selectively secreted different matrix metalloproteinases depending on their metastasis stage and target vasculature, suggesting the possibility of developing drugs that can target each secreted proteinase. We conclude that the 3D tissue tool will be a powerful throughput system for predicting cancer cell responses and time-dependent secretion of molecules in preclinical assays.

Background In this age of antimicrobial resistance, unnecessary use of antibiotics to treat non-bacterial acute respiratory tract infections (ARTIs) and inappropriate use of antibiotics in treating bacterial ARTIs are public health concerns. Purpose Our aim is to identify the pattern of oral antibiotic prescriptions for outpatients with ARTIs in Japan. Methods We analysed health insurance claims data of patients (aged ≤74 years) from 2013 to 2015, to determine the pattern of antibiotic prescriptions for outpatient ARTIs and calculated the proportion of each antibiotic. Results Data on 4.6 million antibiotic prescriptions among 1559394 outpatients with ARTIs were analysed. The most commonly prescribed classes of antibiotics included cephalosporins (41.9%), macrolides (32.8%) and fluoroquinolones (14.7%). The proportion of first-, second- and third-generation cephalosporins was 1.0%, 1.7% and 97.3%, respectively. Fluoroquinolones accounted for a quarter of the prescriptions for ARTIs in patients aged >20 years. In contrast, penicillins accounted for just 8.0% of the total number of antibiotic prescriptions for ARTIs. Conclusions According to clinical guidelines, penicillins are first-line antibiotics against ARTIs. However, third-generation cephalosporins, macrolides and fluoroquinolones are more frequently prescribed in Japan. Although we could not assess the extent to which appropriate antibiotics are selected, our results support the necessity of improving antibiotic choices in the treatment of ARTIs.

Dementia is a major public health concern in ageing societies. Although the population of Japan is among the most aged worldwide, long-term trends in the place of death (PoD) among patients with dementia is unknown. In this Japanese nationwide observational study, we analysed trends in PoD using the data of patients with dementia who were aged ≥65 years and died during 1999–2016. Trends in the crude death rates and PoD frequencies were analysed using the Joinpoint regression model. Changes in these trends were assessed using the Joinpoint regression analysis in which significant change points, the annual percentage change (APC) and average APCs (AAPC) in hospitals, homes, or nursing homes were estimated. During 1999–2016, the number of deaths among patients with dementia increased from 3,235 to 23,757 (total: 182,000). A trend analysis revealed increased mortality rates, with an AAPC of 8.2% among men and 9.3% among women. Most patients with dementia died in the hospital, although the prevalence of hospital deaths decreased (AAPC: -1.0%). Moreover, the prevalence of nursing home deaths increased (AAPC: 5.6%), whereas the prevalence of home deaths decreased (AAPC: -5.8%). These findings support a reconsideration of the end-of-life care provided to patients with dementia.

ObjectivesFall-related mortality among older adults is a major public health issue, especially for ageing societies. This study aimed to investigate current trends in fall-related mortality in Japan using nationwide population-based data covering 1997–2016.DesignWe analysed fall-related deaths among older persons aged ≥65 years using the data provided by the Japanese Ministry of Health, Labour and Welfare.ResultsThe crude and age-standardised mortality rates were calculated per 100 000 persons by stratifying by age (65–74, 75–84 and ≥85 years) and sex. To identify trend changes, a joinpoint regression model was applied by estimating change points and annual percentage change (APC). The total number of fall-related deaths in Japan increased from 5872 in 1997 to 8030 in 2016, of which 78.8% involved persons aged ≥65 years. The younger population (65–74 years) showed continuous and faster-decreasing trends for both men and women. Average APC among men aged ≥75 years did not decrease. Among middle-aged and older women (75–84 and ≥85 years) decreasing trends were observed. Furthermore, the age-adjusted mortality rate of men was approximately twice that of women, and it showed a faster decrease for women.ConclusionsAlthough Japanese healthcare has shown improvement in preventing fall-related deaths over the last two decades, the crude mortality for those aged over 85 years remains high, indicating difficulty in reducing fall-related deaths in the super-aged population. Further investigations to uncover causal factors for falls in older populations are required.

Pancreatic cancer is known for its dismal prognosis despite efforts to improve therapeutic outcome. Recently, cancer nanomedicine, application of nanotechnology to cancer diagnosis and treatment, has gained interest for treatment of pancreatic cancer. The enhanced permeability and retention (EPR) effect that promotes selective accumulation of nanometer‐sized molecules within tumors is the theoretical rationale of treatment. However, it is clear that EPR may be insufficient in pancreatic cancer as a result of stromal barriers within the tumor microenvironment (TME). These limit intratumoral accumulation of macromolecules. The TME and stromal barriers inside it consist of various stromal cell types which interact both with each other and with tumor cells. We are only beginning to understand the complexities of the stromal barriers within the TME and its functional consequences for nanomedicine. Understanding the complex crosstalk between barrier stromal cells is challenging because of the difficulty of modeling pancreatic cancer TME. Here we provide an overview of stromal barriers within the TME. We also describe the preclinical models, both in vivo and in vitro, developed to study them. We furthermore discuss the critical gaps in our understanding, and how we might formulate a better strategy for using nanomedicine against pancreatic cancer.

Angiogenesis is a multi-step process that culminates in vascular maturation whereby nascent vessels stabilize to become functional, and mural cells play an essential role in this process. Recent studies have shown that mural cells in tumors also promote and maintain vascular integrity, with wide-reaching clinical implications including the regulation of tumor growth, metastases, and drug delivery. Various regulatory signaling pathways have been hitherto implicated, but whether regulation of Fas-dependent apoptotic mechanisms is involved has not yet been fully investigated. We first compared endothelial FAS staining in human pancreatic ductal adenocarcinomas and colon carcinomas and show that the latter, characterized by lower mural cell coverage of tumor vasculature, demonstrated higher expression of FAS than the former. Next, in an in vitro coculture system of MS-1 and 10T1/2 cells as endothelial and mural cells respectively, we show that mural cells decreased endothelial Fas expression. Then, in an in vivo model in which C26 colon carcinoma cells were inoculated together with MS-1 cells alone or with the further addition of 10T1/2 cells, we demonstrate that mural cells prevented hemorrhage. Finally, knockdown of endothelial Fas sufficiently recapitulated the protection against hemorrhage seen with the addition of mural cells. These results together suggest that regulation of endothelial Fas signaling is involved in the promotion of vascular integrity by mural cells in tumors.

The survival rate of cardiac arrest patients is less than 10%; therefore, development of a therapeutic strategy that improves their prognosis is necessary. Herein, we searched data collected from medical facilities throughout Japan for drugs that improve the survival rate of cardiac arrest patients. Candidate drugs, which could improve the prognosis of cardiac arrest patients, were extracted using “TargetMine,” a drug discovery tool. We investigated whether the candidate drugs were among the drugs administered within 1 month after cardiac arrest in data of cardiac arrest cases obtained from the Japan Medical Data Center. Logistic regression analysis was performed, with the explanatory variables being the presence or absence of the administration of those candidate drugs that were administered to ≥10 patients and the objective variable being the “survival discharge.” Adjusted odds ratios for survival discharge were calculated using propensity scores for drugs that significantly improved the proportion of survival discharge; the influence of covariates, such as patient background, medical history, and treatment factors, was excluded by the inverse probability-of-treatment weighted method. Using the search strategy, we extracted 165 drugs with vasodilator activity as candidate drugs. Drugs not approved in Japan, oral medicines, and external medicines were excluded. Then, we investigated whether the candidate drugs were administered to the 2,227 cardiac arrest patients included in this study. The results of the logistic regression analysis showed that three (isosorbide dinitrate, nitroglycerin, and nicardipine) of seven drugs that were administered to ≥10 patients showed significant association with improvement in the proportion of survival discharge. Further analyses using propensity scores revealed that the adjusted odds ratios for survival discharge for patients administered isosorbide dinitrate, nitroglycerin, and nicardipine were 3.35, 5.44, and 4.58, respectively. Thus, it can be suggested that isosorbide dinitrate, nitroglycerin, and nicardipine could be novel therapeutic agents for improving the prognosis of cardiac arrest patients.

Social innovation is not only about tangible new products, services, policies, and laws, but also about changes in societal perceptions, values, and norms. In Japan, current policies for older people, including Long-Term Care Insurance, tend to focus on medical and long-term care and other forms of “support” for older adults such as a pension. Naturally, these policies depict older adults as the “beneficiaries,” or the ones in need of support. However, when we look back at pre-modern Japan, it was not always like that. Although older adults did depend on support from family and community members, they also played an active role as a laborer and caretaker as well as providing useful knowledge for their family and community. Moreover, currently, in different areas suffering from a sharp decline in population, such as Okayama prefecture in western Japan, older people are actually playing the role of the supporter for groups of people who are in need, not only the aged population but also other demographics including young children and parents. Based on this historic “tradition” and the present reality, this paper argues that we need to reestablish the image of (at least some) older people as capable of taking a more active and responsible role in society, and position them as such in the new “welfare society” systems in order to replace the conventional “welfare state” model.

Nanomedicine has been heralded as the elusive “magic bullet” of cancer chemotherapeutics ever since the description of the Enhanced Permeability and Retention (EPR) effect in 1986. However, decades of research have often shown a great discrepancy between the salient effects seen in preclinical models and the suboptimal effects seen in clinical trials. This indicates that various obstacles exist within human tumors that impede the delivery and penetration of nanomedicine and that the EPR effect itself may be necessary but not sufficient for an efficacious nanomedicine formulation. Furthermore, these obstacles may be absent or much weaker in often used preclinical models, pointing at the importance of developing novel, clinically relevant preclinical models for testing the efficacy of nanomedicine. It is becoming increasingly clear that the various cellular and extracellular matrix components of the tumor stroma that together consist the tumor microenvironment (TME) play an important role in determining the efficiency of nanomedicine penetration into the tumor. We refer to the impediments that these stromal components of the TME pose to nanomedicine as “stromal barriers”. In this chapter, we review the factors affecting nanomedicine delivery with a particular emphasis on the stromal barriers within the TME. We also review the preclinical models available for testing the efficacy of nanomedicine, and how novel models might be developed to further our understanding of the principles governing nanomedicine delivery and penetration into tumors.

Enhanced permeability in tumours is thought to result from malformed vascular walls with leaky cell-to-cell junctions. This assertion is backed by studies using electron microscopy and polymer casts that show incomplete pericyte coverage of tumour vessels and the presence of intercellular gaps. However, this gives the impression that tumour permeability is static amid a chaotic tumour environment. Using intravital confocal laser scanning microscopy we show that the permeability of tumour blood vessels includes a dynamic phenomenon characterized by vascular bursts followed by brief vigorous outward flow of fluid (named 'eruptions') into the tumour interstitial space. We propose that 'dynamic vents' form transient openings and closings at these leaky blood vessels. These stochastic eruptions may explain the enhanced extravasation of nanoparticles from the tumour blood vessels, and offer insights into the underlying distribution patterns of an administered drug.

The design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid- and polymeric-vesicle-based nanoreactors have some practical limitations. We have succeeded in preparing enzyme-loaded polyion complex vesicles (PICsomes) through a facile protein-loading method. The preservation of enzyme activity was confirmed even after cross-linking of the PICsomes. The cross-linked β-galactosidase-loaded PICsomes (β-gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER-βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4 days after administration of the β-gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme-loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy.