Extracellular Vesicles in Cancer Metastasis: Potential as Therapeutic Targets and Materials.

The vast majority of cancer-related deaths are due to metastasis of the primary tumor that develops years to decades after apparent cures. However, it is difficult to effectively prevent or treat cancer metastasis. Recent studies have shown that communication between cancer cells and surrounding cells enables cancer progression and metastasis. The comprehensive term "extracellular vesicles" (EVs) describes lipid bilayer vesicles that are secreted to outside cells; EVs are well-established mediators of cell-to-cell communication. EVs participate in cancer progression and metastasis by transferring bioactive molecules, such as proteins and RNAs, including microRNAs (miRNAs), between cancer and various cells in local and distant microenvironments. Clinically, EVs functioning as diagnostic biomarkers, therapeutic targets, or even as anticancer drug-delivery vehicles have been emphasized as a result of their unique biological and pathophysiological characteristics. The potential therapeutic effects of EVs in cancer treatment are rapidly emerging and represent a new and important area of research. This review focuses on the therapeutic potential of EVs and discusses their utility for the inhibition of cancer progression, including metastasis.

Cancer cells with high-metastatic potential promote a glycolytic shift in activated fibroblasts.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts and are the major stromal component in various types of malignancies. CAFs often undergo metabolic reprogramming to create an appropriate microenvironment for cancer progression. However, it remains unclear whether the metastatic properties of cancer cells affect aerobic glycolysis in stromal cells. Here, we show that gastric cancer (GC) cells with high metastatic potential strongly promote the metabolic switch from oxidative phosphorylation to aerobic glycolysis in fibroblasts. Transcriptome analysis showed that the expression of glycolysis-related genes, such as LDHA and ENO2, significantly changed in fibroblasts when they were cocultured with cancer cells with high metastatic potential compared to fibroblasts incubated with cancer cells with low metastatic potential. Glucose uptake, lactate production and oxygen consumption in fibroblasts were changed by coculture with GC cells with high metastatic potential. Thus, metabolic reprogramming in CAFs may reflect the metastatic properties of GC cells.

Quercetin Inhibits Lef1 and Resensitizes Docetaxel-Resistant Breast Cancer Cells.

Drug resistance is a major problem for breast cancer patients. Docetaxel is an anti-mitotic agent that serves as first line of treatment in metastatic breast cancer, however it is susceptible to cellular drug resistance. Drug-resistant cells are able to spread during treatment, leading to treatment failure and eventually metastasis, which remains the main cause for cancer-associated death. In previous studies, we used single-cell technologies and identified a set of genes that exhibit increased expression in drug-resistant cells, and they are mainly regulated by Lef1. Furthermore, upregulating Lef1 in parental cells caused them to become drug resistant. Therefore, we hypothesized that inhibiting Lef1 could resensitize cells to docetaxel. Here, we confirmed that Lef1 inhibition, especially on treatment with the small molecule quercetin, decreased the expression of Lef1 and resensitized cells to docetaxel. Our results demonstrate that Lef1 inhibition also downregulated ABCG2, Vim, and Cav1 expression and equally decreased Smad-dependent TGF-ß signaling pathway activation. Likewise, these two molecules worked in a synergetic manner, greatly reducing the viability of drug-resistant cells. Prior studies in phase I clinical trials have already shown that quercetin can be safely administered to patients. Therefore, the use of quercetin as an adjuvant treatment in addition to docetaxel for the treatment of breast cancer may be a promising therapeutic approach.

Cancer extracellular vesicles contribute to stromal heterogeneity by inducing chemokines in cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs), one of the major components of a tumour microenvironment, comprise heterogeneous populations involved in tumour progression. However, it remains obscure how CAF heterogeneity is governed by cancer cells. Here, we show that cancer extracellular vesicles (EVs) induce a series of chemokines in activated fibroblasts and contribute to the formation of the heterogeneity. In a xenograft model of diffuse-type gastric cancer, we showed two distinct fibroblast subpopulations with alpha-smooth muscle actin (α-SMA) expression or chemokine expression. MicroRNAs (miRNAs) profiling of the EVs and the transfection experiment suggested that several miRNAs played a role in the induction of chemokines such as CXCL1 and CXCL8 in fibroblasts, but not for the myofibroblastic differentiation. Clinically, aberrant activation of CXCL1 and CXCL8 in CAFs correlated with poorer survival in gastric cancer patients. Thus, this link between chemokine expression in CAFs and tumour progression may provide novel targets for anticancer therapy.

Exploiting the message from cancer: the diagnostic value of extracellular vesicles for clinical applications.

Liquid biopsy is indispensable for the resolution of current medical issues, such as the cost of developing new drugs and predicting responses of patients to drugs. In this sense, not only the technology for liquid biopsy but also the target biomolecules for biomarkers need to be identified. Extracellular vesicles (EVs), which contain various proteins, including membrane-bound proteins, and RNAs, including mRNA and long/short noncoding RNAs, have emerged as ideal targets for liquid biopsy. These complex biomolecules are covered by a lipid bilayer, which can protect them from degradation. In this review, we review current topics regarding EVs as cancer biomarkers and introduce technologies used for these recently emerged biomolecules.

Cross-talk between cancer cells and their neighbors via miRNA in extracellular vesicles: an emerging player in cancer metastasis.

Cancer metastasis is the major cause of mortality in cancer cases and is responsible for cancer deaths. It is known that cancer cells communicate with surrounding microenvironmental cells, such as fibroblast cells, immune cells, and endothelial cells, to create a cancer microenvironment for their progression. Extracellular vesicles (EVs) are small vesicles that can be secreted by most types of cells and play an important role in cell-to-cell communications via transferring bioactive cargos, including variable RNAs, such as microRNAs (miRNAs), to recipient cells. miRNAs are a class of small noncoding RNAs that posttranscriptionally regulate gene expression. The transfer of them to recipient cells influences the metastatic process of primary tumors. In this review, we summarize the function of miRNAs packaged in EVs in cancer metastasis and discuss the clinical utility of miRNAs in EVs.

Macular vessel reduction as predictor for recurrence of macular oedema requiring repeat intravitreal ranibizumab injection in eyes with branch retinal vein occlusion.

AIM: To determine whether there are factors that can predict the frequency of recurrences of macular oedema associated with branch retinal vein occlusion (BRVO). METHODS: We reviewed the medical records of 31 eyes with treatment-naïve macular oedema associated with BRVO. All eyes received an intravitreal ranibizumab (IVR) injection and were followed with a pro re nata protocol for at least 12 months. A reinjection of ranibizmab was performed when the central foveal thickness was ≥300 µm. At 1 month after IVR injection, the macular vessel reduction was calculated by comparing the vessel density in the optical coherence tomography angiography in the BRVO involved half to that in the non-involved half. RESULTS: The mean visual acuity improved from 0.35±0.27 logarithm of the minimal angle of resolution (logMAR) units (20/45; Snellen) at initial visit to 0.06±0.15 logMAR units (20/23) at 12 months (p<0.0001). During 12 months, the mean number of IVR injections was 3.8±1.8. Multivariate regression analysis showed that a greater macular vessel reduction at 1 month after initial IVR injection was significantly a negative predictor of frequency of IVR injections (ß=-0.5065, p=0.0082). The visual acuity and the central foveal thickness at the initial visit or at 1 month after initial IVR injection were not predictive factors for frequency of IVR injections. CONCLUSIONS: Patients with BRVO with a large macular vessel reduction at 1 month after an initial IVR injection have fewer recurrences and thus lower frequency of IVR injections during 12 months.

Screening of microRNAs for a repressor of hepatitis B virus replication.

BACKGROUND: Hepatitis B virus (HBV) infection is a leading cause of persistent liver diseases, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Since deregulation of microRNA (miRNA) expression by HBV infection contributes to enhanced viral replication and pathogenesis, modulation of miRNA activity can be a novel therapeutic approach towards HBV eradication. As the effects of the vast majority of miRNAs on HBV replication have not been empirically investigated, here, we aim to identify novel therapeutic targets that have a strong antiviral effect on HBV. METHODS: HepG2-hNTCP-C4 cells were infected with HBV, and then were individually transfected with the library mimics of 2048 miRNAs. To assess the amount of intracellular and extracellular DNA and HBsAg, qPCR and ELISA were performed respectively. RESULTS: From miRNA library screening, we identified 39 miRNAs as candidate repressors of HBV replication. Among them, 9 miRNAs, including miR-204, strongly decreased both HBV DNA and HBsAg in culture supernatant of HepG2-hNTCP-C4 cells. Furthermore, we also showed that inhibition of Rab22a, one of the targets of miR-204, also suppressed intracellular and extracellular HBV DNA expression in HepG2.2.15.7 cells. CONCLUSIONS: Our findings contribute to the understanding of the roles of miRNAs underlying HBV replication and show the possibility of developing a novel strategy for miRNA-mediated HBV treatment.

Optical coherence tomographic predictor of retinal non-perfused areas in eyes with macular oedema associated with retinal vein occlusion.

AIM: To determine whether the low reflective spaces in the retinal nerve fibre layer (RNFL) in the optical coherence tomographic (OCT) images are related to the presence of the retinal non-perfused areas in eyes with macular oedema associated with a retinal vein occlusion (RVO). METHODS: We reviewed the medical records of 97 eyes with macular oedema associated with RVO. At the initial visit, eyes with macular oedema were classified into those with and those without low reflective spaces in the RNFL in the OCT images. In the fluorescein angiographic (FA) images, the eyes with more than one disc diameter of retinal non-perfusion in the area of OCT vertical scan were classified as having retinal non-perfused areas. RESULTS: At the initial visit, low reflective spaces were observed in the RNFL in 19 of the 97 eyes with macular oedema associated with a RVO (19.6%). The percentage of eyes with a retinal non-perfused area in the FA was 84.2% in the eyes with low reflective space group but only 14.1% in those without low reflective spaces. The presence of low reflective spaces in the RNFL was significantly correlated with the presence of non-perfused areas (p<0.0001). CONCLUSION: The presence of low reflective spaces in the RNFL in the OCT images can predict the presence of retinal non-perfused areas in eyes with macular oedema associated with a RVO.

Potential anti-osteoporotic effects of herbal extracts on osteoclasts, osteoblasts and chondrocytes in vitro.

BACKGROUND: Osteoporosis (OP) is one of the most serious diseases in the modern world, and OP patients frequently suffer from fragility fractures in the hip, spine and wrist, resulting in a limited quality of life. Although bisphosphonates (BPs) are the most effective class of anti-bone-resorptive drugs currently available and the most commonly prescribed for the clinical treatment of OP, they are known to cause serious side effects such as bisphosphonate-related osteonecrosis of the jaw. Novel therapeutic materials that can replace the use of BPs have therefore been developed. METHODS: We commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only in OP, but also in oral and skeletal diseases. In the present study, we report on 3 Chinese medical herbal extracts from the root barks of Melia azedarach, Corydalis turtschaninovii, and Cynanchum atratum. RESULTS: All of these extracts inhibited osteoclast proliferation and induced apoptosis by up-regulation of caspase activity and increase of mitochondrial pro-apoptotic proteins expression. Furthermore, the extracts enhanced differentiation, but did not affect proliferation of both osteoblasts and chondrocytes. The osteo-inducible effect was also observed in cultured primary bone marrow cells. CONCLUSIONS: Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects in OP. In this study, we elucidate the potency of these herbal extracts as novel candidates for OP therapy.

Efficacy and retention times of intravitreal triamcinolone acetonide for macular edema.

PURPOSE: To evaluate the retention of intravitreal triamcinolone acetonide (TA) particles and the efficacy of TA therapy for patients with cystoid macular edema in branch retinal vein occlusion (BRVO) or diabetic macular edema (DME). We monitored the TA particles until absorption from the vitreous cavity was complete. The correlation between the intravitreal retention time of TA and its efficacy was evaluated based on central macular thickness (CMT). RESULTS: The intravitreal TA retention time was a mean 141.8 +/- 139.6 days in BRVO patients and 114.5 +/- 59.6 days in DME patients. Patient age and retention time were negatively correlated (r = -0.46; P = 0.013). At 6 months posttreatment the mean CMT decreased from 544.1 +/- 143.7 to 322.4 +/- 131.9 mum in BRVO patients and from 454.5 +/- 119.0 to 371.2 +/- 209.4 mum in DME patients. Retention time and CMT reduction were positively correlated in BRVO patient (r = 0.56, P = 0.02) but not in DME patients (P = 0.06). CONCLUSIONS: Intravitreal TA reduced the CMT in BRVO and DME patients over 6 months. The retention time was longer in younger individuals. The efficacy of the therapy depended on the intravitreal TA retention time observed clinically in BRVO patients. Biomicroscopic examination of intravitreal TA is useful for evaluation of its efficacy.