miR-1246 in tumor extracellular vesicles promotes metastasis via increased tumor cell adhesion and endothelial cell barrier destruction

BackgroundTumor blood vessels play a key role in tumor metastasis. We have previously reported that tumor endothelial cells (TECs) exhibit abnormalities compared to normal endothelial cells. However, it is unclear how TECs acquire these abnormalities. Tumor cells secrete extracellular vesicles (EVs) to create a suitable environment for themselves. We have previously identified miR-1246 to be more abundant in high metastatic melanoma EVs than in low metastatic melanoma EVs. In the current study, we focused on miR-1246 as primarily responsible for acquiring abnormalities in TECs and examined whether the alteration of endothelial cell (EC) character by miR-1246 promotes cancer metastasis.MethodsWe analyzed the effect of miR-1246 in metastatic melanoma, A375SM-EVs, in vivo metastasis. The role of tumor EV-miR-1246 in the adhesion between ECs and tumor cells and the EC barrier was addressed. Changes in the expression of adhesion molecule and endothelial permeability were examined.ResultsIntravenous administration of A375SM-EVs induced tumor cell colonization in the lung resulting in lung metastasis. In contrast, miR-1246 knockdown in A375SM decreased lung metastasis in vivo. miR-1246 transfection in ECs increased the expression of adhesion molecule ICAM-1 via activation of STAT3, followed by increased tumor cell adhesion to ECs. Furthermore, the expression of VE-Cadherin was downregulated in miR-1246 overexpressed EC. A375SM-EV treatment enhanced endothelial permeability. VE-Cadherin was validated as the potential target gene of miR-1246 via the target gene prediction database and 3′ UTR assay.ConclusionmiR-1246 in high metastatic tumor EVs promotes lung metastasis by inducing the adhesion of tumor cells to ECs and destroying the EC barrier

Tumor-Derived Microvesicles Induce Proangiogenic Phenotype in Endothelial Cells via Endocytosis

Background: Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear. Methodology/Principal Findings: Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis. Conclusion: We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment

Application of "Dredging Method" for the patients with odontogenic keratocyst.

When conservative treatment such as enucleation is applied, odontogenic keratocyst (OKC) is a well-known jaw lesion with a high recurrence rate. Other treatment modalities aimed at preventing recurrence have been used for managing OKCs. In our institute, an alternative conservative approach, known as the dredging method, is applied in OKC treatment. This paper describes and reports the characteristics and outcomes of conservative treatments involving the dredging method and enucleation in the management of patients with OKC. Seventy-four patients with OKC or keratocystic odontogenic tumor (KCOT) presented to Oral and Maxillofacial Surgery at Hokkaido University Hospital between 1983 and 2017. They were treated and followed for more than 12 months. The patients’ median age was 40 years (range 7‒72 years) and the median longitudinal diameter of the lesion was 30 mm (range 7‒128 mm). A total of 14 cysts (18.9%) were multilocular, as determined via radiographic evaluation. The surgical intervention consisted of enucleation alone in 17 cases (22.3%), deflation followed by enucleation in seven cases (9.5 %), and the dredging method in 50 cases. Statistical significance associated with the size ( P = 0.01) and locularity ( P = 0.02) of the lesions was found between the cases treated with enucleation versus the dredging method. The median duration of follow-up was 40 months (range 12 to 405 months). Recurrence occurred in 9 of 74 cases (12.2%), including 2 of 17 cases (11.8%) treated with enucleation and 7 of 57 cases (12.3 %) treated wit h the dredging method. The recurrence period ranged from 12 to 131 months. All recurrence cases were managed by enucleation. There was no correlation between recurrence and patient sex or age, lesion size or locularity, the presence of a daughter cyst, or surgical approach. These results suggest that the dredging method is a s uccessful conservative treatment option for large, multilocular OKCs. Long-term regular follow-up is essential to identify and manage recurrent cases

Synergistic Enhancement of Cellular Uptake With CD44-Expressing Malignant Pleural Mesothelioma by Combining Cationic Liposome and Hyaluronic Acid-Lipid Conjugate

Malignant pleural mesothelioma (MPM) is a highly aggressive form of cancer, with a median survival of less than 1 year. It is well known that the hyaluronan (HA) receptor CD44 is highly expressed by MPM cells and is reported to be correlated with a poor prognosis. We herein report on the development of a new type if drug delivery system against CD44 that involves the use of lipid nanoparticles (LNPs) equipped with a new type of HA derivative. In this study, we evaluated HA-lipid conjugation (HAL) via the end of the HA molecule through reductive amination, a process that allowed the carboxylate group to remain intact. As a result, the HAL-modified LNP appears to be a potent nanoparticle for dealing with MPM. Surprisingly, the use of a combination of a cationic lipid and HAL had a synergistic effect on cellular uptake in MPM and consequently permitted an anti-cancer drug such as cis-diamminedichloro-platinum(II) (CDDP). Intrapleural injection of CDDP-loaded HAL-LNP (1.5 mg/kg as CDDP) per week significantly suppressed the progression of this type of cancer in an MPM orthotopic model. These results suggest that HAL-modified LNP represents a potent delivery system for MPM cells that express high levels of CD44. (c) 2019 American Pharmacists Association (R). Published by Elsevier Inc. All rights reserved

Contribution of Tumor Endothelial Cells in Cancer Progression

Tumor progression depends on the process of angiogenesis, which is the formation of new blood vessels. These newly formed blood vessels supply oxygen and nutrients to the tumor, supporting its progression and providing a gateway for tumor metastasis. Tumor angiogenesis is regulated by the balance between angiogenic activators and inhibitors within the tumor microenvironment. Because the newly formed tumor blood vessels originate from preexisting normal vessels, tumor blood vessels, and tumor endothelial cells (TECs) have historically been considered to be the same as normal blood vessels and endothelial cells; however, evidence of TECs’ distinctive abnormal phenotypes has increased. In addition, it has been revealed that TECs constitute a heterogeneous population. Thus, TECs that line tumor blood vessels are important targets in cancer therapy. We have previously reported that TECs induce cancer metastasis. In this review, we describe recent studies on TEC abnormalities related to cancer progression to provide insight into new anticancer therapies

Application of POLARIC (TM) fluorophores in an in vivo tumor model

Fluorescent and luminescent tools are commonly used to study the dynamics of cancer progression and metastases in real-time. Fluorophores have become essential tools to study biological events. However, few can sustain fluorescence long enough during long-term studies. In the present study, we focused on a series of new amphiphilic fluorophores known as POLARIC (TM), which emit strong fluorescence in lipid bilayers and can be readily modified using the Suzuki-Miyaura cross-coupling reaction. Appropriate chemical modifications of substituent groups can improve target-site specificity, reduce cytotoxicity and prolong emission. Therefore, in contrast to conventional fluorescent probes, these fluorophores show promise for long-term monitoring of biological processes. In the present study, we conducted long-term observations of tumor growth and metastasis using a POLARIC derivative as a novel fluorescent probe. For this purpose, we studied the metastatic melanoma cell line A375-SM, which proliferates at a high rate. We compared the characteristics of the POLARIC probe with the commercially available fluorescent dye PKH26 and fluorescent protein mRFP1. A375-SM cells were labeled with these fluorescent probes and orthotopically implanted into nude mice. The fluorescence emitted by POLARIC was detected more than five weeks after implantation without causing detectable harmful effects on tumor growth. By contrast, fluorescence of cells labeled with PKH26 could not be detected at this same time. Furthermore, POLARIC-, but not PKH26-labeled cells, were also detected in lung metastases. These results indicate that labeling cells with POLARIC fluorophores can significantly extend the time course of in vivo studies on tumor cell growth

Comparison of characteristics of mouse immortalized normal endothelial cells, MS1 and primary cultured endothelial cells.

Tumor blood vessels support the progression of tumors by providing nutrition and oxygen required for growth. By acting as gatekeepers, they allow the metastasis of tumors to secondary locations. An important strategy in cancer therapy has been to target tumor blood vessels consequently inhibiting tumor angiogenesis. To date, antiangiogenic therapy being employed for cancer treatment have yielded profoundly good results. However, it has been shown that current antiangiogenic drugs have several problems, such as adverse side effects and drug resistance. Tumor endothelial cells (TEC), which line the inner layer of blood vessels of the tumor stromal tissue, are the main targets of the antiangiogenic therapies. TEC have been reported to differ significantly from endothelial cells resident in normal blood vessels. These differences provide a window through which TEC can be targeted solely with little or no impact on normal endothelial cells (NEC). Currently, as part of new antiangiogenic drug discovery processes, cell-based screening is being performed using thousands of small chemical compounds. For the success of such screening purposes, there is a need to obtain the right kind of cells and in adequate quantities. Primary–cultured endothelial cells isolated from murine / human blood vessels are the preferred choice. However, maintenance of the primary-cultured endothelial cells is costly and overtime these cells become senescent and perish. As a result, MS1, SV40 immortalized islet-derived endothelial cells, have been used in place of the primary-cultured cells. MS1 is commercially available with comparatively cheaper cell culture requirements. 　In this study, we compared the characteristics of MS1 and primary-cultured endothelial cells ; NEC and TEC to investigate the possibility of using MS1 cells for chemical screening in search of a new antiangiogenic drug. MS1 cells proliferate faster compared to TEC and upregulated the mRNA expressions of CD133 and Sca-1 genes. However, mRNA expression of most of the other genes, which were upregulated in TEC compared to NEC, were also expressed at lower levels in the MS1 cells. Furthermore, MS1 migrated at a slower rate and did not form tubes on matrigel, as opposed to the function of TEC. In conclusion, MS1 did not completely resemble NEC, nor TEC in function and gene expression. It is suggested that for chemical screening, primary-cultured TEC and the corresponding NEC would be a more ideal choice of cells

ROS enhance angiogenic properties via regulation of NRF2 in tumor endothelial cells

Reactive oxygen species (ROS) are unstable molecules that activate oxidative stress. Because of the insufficient blood flow in tumors, the tumor microenvironment is often exposed to hypoxic condition and nutrient deprivation, which induces ROS accumulation. We isolated tumor endothelial cells (TECs) and found that they have various abnormalities, although the underlying mechanisms are not fully understood. Here we showed that ROS were accumulated in tumor blood vessels and ROS enhanced TEC migration with upregulation of several angiogenesis related gene expressions. It was also demonstrated that these genes were upregulated by regulation of Nuclear factor erythroid 2-related factor 2 (NRF2). Among these genes, we focused on Biglycan, a small leucine-rich proteoglycan. Inhibition of Toll-like receptors 2 and 4, known BIGLYCAN (BGN) receptors, cancelled the TEC motility stimulated by ROS. ROS inhibited NRF2 expression in TECs but not in NECs, and NRF2 inhibited phosphorylation of SMAD2/3, which activates transcription of BGN. These results indicated that ROS-induced BGN caused the pro-angiogenic phenotype in TECs via NRF2 dysregulation