Uterine leiomyosarcoma cell-derived extracellular vesicles induce the formation of cancer-associated fibroblasts.

OBJECTIVE: Uterine leiomyosarcoma (ULMS) is a rare malignant tumor, which is aggressive, and has a poor prognosis even during its early stages. Extracellular vesicles (EVs) carry cargo, such as microRNAs (miRNAs), which are involved in intercellular communication in the tumor microenvironment and other processes. Because there are no studies on EV-related miRNAs in ULMS, we identified EV-related miRNAs in ULMS and examined their function. METHODS: Small EVs (sEVs) and medium/large EVs (m/lEVs) were extracted from ULMS cells by ultracentrifugation and their basic characteristics were evaluated. Then, small RNA sequencing was done to obtain EV-related miRNA profiles. Next, miRNA expression levels in sera and tissues of ULMS patients were compared with those of myoma patients. RESULTS: miR-654-3p and miR-369-3p were indicated to be highly expressed in both sera and tissues of ULMS patients. These two miRNAs are also highly expressed in ULMS cell lines and ULMS-derived EVs. Some cancer-associated fibroblast (CAF) markers were increased when fibroblasts were treated with ULMS-derived EVs. Furthermore, fibroblasts took up EVs derived from ULMS as determined by confocal laser microscopy. In addition, the transfection of the two candidate miRNAs into fibroblasts significantly increased some CAF markers, particularly ACTA2. CONCLUSION: miR-654-3p and miR-369-3p are highly expressed in ULMS-derived EVs, indicating that these EV-related miRNAs induce the formation of cancer-associated fibroblasts.

Small extracellular vesicles in follicular fluids for predicting reproductive outcomes in assisted reproductive technology.

BACKGROUND: Assisted reproductive technology accounts for an increasing proportion of infertility treatments, and assessments to predict clinical pregnancy outcomes are desired. Extracellular vesicles exist in follicular fluid, and small non coding RNAs in extracellular vesicles underline the possibility of reflecting pregnancy potential. METHODS: Follicular fluid samples are collected from 20 ovarian follicles of 15 infertile patients undergoing assisted reproductive technology. Extracellular vesicles are isolated by serial centrifugation and small RNA sequencing is performed to investigate the profiles of microRNAs and P-element-induced wimpy testis-interacting RNAs. RESULTS: Small extracellular vesicles with a size range of approximately 100 nm are successfully isolated, and the small non coding RNA profiles of pregnant samples (n = 8) are different from those of non-pregnant samples (n = 12). Fourteen dysregulated small non coding RNAs are selected to identify the independent candidates [mean read count >100, area under the curve >0.8]. Among them, we find that a specific combination of small non coding RNAs (miR-16-2-3p, miR-378a-3p, and miR-483-5p) can predict the pregnant samples more precisely using a receiver operating characteristics curves analysis (area under the curve: 0.96). Furthermore, even in the same patients, the three microRNAs are differentially expressed between pregnant and non-pregnant samples. CONCLUSIONS: Our results demonstrate that small non coding RNAs derived from small extracellular vesicles in follicular fluid can be potential non-invasive biomarkers for predicting pregnancy, leading to their probable application in assisted reproductive technology. Further large-scale studies are required to validate the clinical usefulness of these small non coding RNAs.

Assisted reproductive technologies (ART) are medical procedures used to address infertility. Follicular fluid (FF)is a liquid that surrounds the immature egg cells (oocytes) and provides hormones and nutrients necessary for their maturation and eventual development into embryos. We analyzed genetic components within the FF as a potential predictor of reproductive outcomes following ART. Here, we show that a specific combination of genetic elements produced by the FF in successful pregnancies did not occur in unsuccessful pregnancies. Our findings may help to provide a non-invasive approach to determining reproductive outcomes during ART procedures.

Serum miRNA as a predictive biomarker for ovarian reserve after endometrioma-cystectomy.

Ovarian endometrioma (OE) is a common gynecological disease that is often treated with surgery and hormonal treatment. However, ovarian cystectomy can impair the ovarian reserve (OR). Previously, we showed that perioperative administration of dienogest (DNG) is an effective option for OR preservation. However, there were differences in the extent of OR preservation among patients following perioperative DNG treatment. In the current study, we performed a global examination of serum microRNAs (miRNAs) to identify accurate biomarkers that predict post-operative restoration of OR following perioperative DNG treatment. We also sought to identify specific miRNAs related to the anti-Müllerian hormone (AMH). miRNA sequencing was performed on serum samples obtained from twenty-seven patients who received perioperative DNG treatment. Candidate miRNAs were selected by comparing patients whose ORs were restored postoperatively (responder group, n = 7) with those whose ORs were not (non-responder group, n = 7). miR-370-3p and miR-1307-3p were significantly upregulated in the responder group, whereas miR-27b-3p was upregulated in the non-responder group. The pretreatment value of each miRNA could predict DNG responsiveness for OR following ovarian cystectomy (area under the curve [AUC] > 0.8). The quantitative polymerase chain reaction (qPCR) revealed only miR-1307-3p was found to be significantly upregulated in the responder group (P < 0.05). In addition, we identified miR-139-3p, miR-140-3p, and miR-629-5p as AMH-associated miRNAs. The transition of AMH showed a correlation with miR-139-3p (P < 0.05, r = -0.76). The miRNAs identified herein represent potential serum biomarkers of clinical value in predicting OR prior to DNG treatment.

Spatial exosome analysis using cellulose nanofiber sheets reveals the location heterogeneity of extracellular vesicles.

Extracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 µL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

Small Extracellular Vesicles from adipose-derived stem cells suppress cell proliferation by delivering the let-7 family of microRNAs in ovarian cancer.

INTRODUCTION: Ovarian cancer is the leading cause of death among women with gynecological cancer, and novel treatment options are urgently needed. Extracellular vesicles (EVs), including exosomes, may be one of the most promising therapeutic tools for various diseases. In this study, we aimed to investigate the therapeutic effects of adipose-derived stem cell-derived EVs (ADSC-EVs) on ovarian cancer cell lines. MATERIALS AND METHODS: ADSCs and the ovarian cancer cell lines SKOV3 and OV90 were used for analysis. ADSC-EVs were isolated through ultracentrifugation and validated using a cryotransmission electron microscope, nanoparticle tracking analysis, and western blotting. Then, the effect of ADSC-EVs on ovarian cancer cells was investigated using IncuCyte and microRNA sequencing. Moreover, the potential functions of miRNAs were evaluated by gain-of function analysis and in silico analysis. RESULTS: ADSC-EVs suppressed SKOV3 and OV90 cell proliferation. In particular, small EVs (sEVs) from ADSCs exhibited a stronger antitumor effect than ADSC-medium/large EVs (m/lEVs). Comparison of the miRNA profiles between ADSC-sEVs and ADSC-m/lEVs, along with downstream pathway analysis, suggested the involvement of the let-7 family. Overexpression of hsa-let-7b-5p and hsa-let-7e-5p significantly suppressed the proliferation of SKOV3 cells. In silico analysis revealed that four potential target genes of hsa-let-7b-5p and hsa-let-7e-5p were significantly associated with the prognoses of the patients. CONCLUSION: ADSC-sEVs had a stronger antitumor effect than ADSC-m/lEVs. Hsa-let-7b-5p and hsa-let-7e-5p, which are highly abundant in ADSC-sEVs, suppressed cell proliferation. These findings may open up new possibilities for therapeutic approaches using ADSC-sEVs.

Identifying high-grade serous ovarian carcinoma-specific extracellular vesicles by polyketone-coated nanowires.

Cancer cell-derived extracellular vesicles (EVs) have unique protein profiles, making them promising targets as disease biomarkers. High-grade serous ovarian carcinoma (HGSOC) is the deadly subtype of epithelial ovarian cancer, and we aimed to identify HGSOC-specific membrane proteins. Small EVs (sEVs) and medium/large EVs (m/lEVs) from cell lines or patient serum and ascites were analyzed by LC-MS/MS, revealing that both EV subtypes had unique proteomic characteristics. Multivalidation steps identified FRα, Claudin-3, and TACSTD2 as HGSOC-specific sEV proteins, but m/lEV-associated candidates were not identified. In addition, for using a simple-to-use microfluidic device for EV isolation, polyketone-coated nanowires (pNWs) were developed, which efficiently purify sEVs from biofluids. Multiplexed array assays of sEVs isolated by pNW showed specific detectability in cancer patients and predicted clinical status. In summary, the HGSOC-specific marker detection by pNW are a promising platform as clinical biomarkers, and these insights provide detailed proteomic aspects of diverse EVs in HGSOC patients.

Downregulation of miR­10b­5p facilitates the proliferation of uterine leiomyosarcoma cells: A microRNA sequencing­based approach.

Uterine leiomyosarcoma (ULMS) is one of the most aggressive gynecological malignancies. In addition, the molecular background of ULMS has not been fully elucidated due to its low incidence. Therefore, no effective treatment strategies have been established based on its molecular background. The present study aimed to investigate the roles of microRNAs (miRNAs/miRs) in the development of ULMS. Comprehensive miRNA sequencing was performed using six ULMS and three myoma samples, and revealed 53 and 11 significantly upregulated and downregulated miRNAs, respectively. One of the most abundant miRNAs in myoma samples was miR­10b­5p. The mean normalized read count of miR­10b­5p was 93,650 reads in myoma, but only 27,903 reads in ULMS. Subsequently, to investigate the roles of miR­10b­5p, gain­of­function analysis was performed using SK­UT­1 and SK­LMS­1 cell lines. The overexpression of miR­10b­5p suppressed cell proliferation and reduced the number of colonies. Moreover, miR­10b­5p increased the number of cells in the G1 phase. In conclusion, tumor­suppressive miR­10b­5p was significantly downregulated in ULMS compared with in myoma; thus, miR­10b­5p may serve a specific role in sarcoma progression.

Novel therapeutic strategies targeting UCP2 in uterine leiomyosarcoma.

Uterine leiomyosarcoma (ULMS) is a malignant stromal tumor arising from the myometrium with a poor prognosis and very limited response to current chemotherapy. This study aimed to identify novel targets for ULMS through a three-step screening process using a chemical library consisting of 1271 Food and Drug Administration-approved drugs. First, we evaluated their inhibitory effects on ULMS cells and identified four candidates: proscillaridin A, lanatoside C, floxuridine, and digoxin. Then, we subcutaneously or orthotopically transplanted SK-UT-1 cells into mice to establish mouse models. In vivo analyses showed that proscillaridin A and lanatoside C exerted a superior antitumor effect. The results of mRNA sequencing showed that uncoupling protein 2 (UCP2) was suppressed in the sirtuin signaling pathway, increasing reactive oxygen species (ROS) and inducing cell death. Moreover, the downregulation of UCP2 induced ROS and suppressed ULMS cell growth. Furthermore, analyses using clinical samples showed that UCP2 expression was significantly upregulated in ULMS tissues than in myoma tissues both at the RNA and protein levels. These findings suggested that UCP2 is a potential therapeutic target and can contribute to the development of novel therapeutic strategies in patients with ULMS.

Drug library screening identifies histone deacetylase inhibition as a novel therapeutic strategy for choriocarcinoma.

BACKGROUND: Choriocarcinoma is a rare and aggressive gynecological malignancy. The standard treatment is systemic chemotherapy as choriocarcinoma exhibits high chemosensitivity. However, refractory choriocarcinoma exhibits chemoresistance; thus, the prognosis remains very poor. This study aimed to identify novel therapeutic agents for choriocarcinoma by utilizing a drug repositioning strategy. METHODS: Three choriocarcinoma cell lines (JAR, JEG-3, and BeWo) and a human extravillous trophoblast cell line (HTR-8/SVneo) were used for the analyses. The growth inhibitory effects of 1,271 FDA-approved compounds were evaluated in vitro screening assays and selected drugs were tested in tumor-bearing mice. Functional analyses of drug effects were performed based on RNA sequencing. RESULTS: Muti-step screening identified vorinostat, camptothecin (S, +), topotecan, proscillaridin A, and digoxin as exhibiting an anti-cancer effect in choriocarcinoma cells. Vorinostat, a histone deacetylase inhibitor, was selected as a promising candidate for validation and the IC50 values for choriocarcinoma cells were approximately 1 µM. RNA sequencing and subsequent pathway analysis revealed that the ferroptosis pathway was likely implicated, and key ferroptosis-related genes (i.e., GPX4, NRF2, and SLC3A2) were downregulated following vorinostat treatment. Furthermore, vorinostat repressed tumor growth and downregulated the expression of GPX4 and NRF2 in JAR cell-bearing mice model. CONCLUSION: Vorinostat, a clinically approved drug for the treatment of advanced primary cutaneous T-cell lymphoma, showed a remarkable anticancer effect both in vitro and in vivo by regulating the expression of ferroptosis-related genes. Therefore, vorinostat may be an effective therapeutic candidate for patients with choriocarcinoma.

Evaluation of the Geenius HIV 1/2 confirmatory assay for HIV-2 samples isolated in Japan.

BACKGROUND: Although the number of HIV-2-infected individuals is quite low in Japan, at least three groups of HIV-2 (A, B and CRF01_AB) have been detected thus far. In particular, CRF01_AB HIV-2 cases have been found only in limited areas, Cote d'Ivoire and Japan. Here, we demonstrate that Geenius HIV 1/2 Confirmatory Assay (Geenius, Bio-Rad Laboratories) is able to detect HIV-2 samples, including groups A, B and CRF01_AB, isolated in Japan. STUDY DESIGN: A total of 57 plasma samples, including three panels (Ⅰ: HIV-2-positive samples [n=9], Ⅱ: HIV-1 infection with HIV-2 antibody cross-reactivity samples [n=37], and Ⅲ: HIV negative with biological false-positive HIV-2 samples [n=11]) were tested by Geenius. RESULTS: Geenius determined Panel I to be "HIV-2 positive with/without HIV-1 cross-reactivity (n=4, respectively)", including HIV-2 group A and CRF01_AB. In the case with HIV-2 group B, all bands were detected, resulting in a Geenius interpretation of "HIV positive untypable". Geenius classified Panels II and III as "HIV-1 positive (n=37)" or "HIV negative (n=9)", "HIV indeterminate (n=1)" and "HIV-2 indeterminate (n=1)", suggesting 95.8% HIV-2 differentiation by Geenius. CONCLUSIONS: With Geenius, there were fewer false-positives for HIV-1/-2 negativity and fewer cross-reactions with HIV-2 among HIV-1-positive samples. Additionally, the assay could detect HIV-2 genetic group CRF01_AB. Geenius can be expected to be a useful diagnostic tool that is an alternative to conventional Western blotting.

Discovery of 4-oxoquinolines, a new chemical class of anti-HIV-1 compounds.

Antiretroviral therapy (ART) against HIV-1 infection offers the promise of controlling disease progression and prolonging the survival of HIV-1-infected patients. However, even the most potent ART regimens available today cannot cure HIV-1. Because patients will be exposed to ART for many years, physicians and researchers must anticipate the emergence of drug-resistant HIV-1, potential adverse effects of the current drugs, and need for future drug development. In this study, we screened a small-molecule compound library using cell-based anti-HIV-1 assays and discovered a series of novel anti-HIV-1 compounds, 4-oxoquinolines. These compounds exhibited potent anti-HIV-1 activity (EC50 < 0.1 µM) with high selectivity indexes (CC50/EC50 > 2500) and favorable pharmacokinetic profiles in mice. Surprisingly, our novel compounds have a chemical backbone similar to the clinically used integrase (IN) strand transfer inhibitor (INSTI) elvitegravir, although they lack the crucial 3-carboxylate moiety needed for the common INSTI diketo motif. Indeed, the new 4-oxoquinoline derivatives have no detectable INSTI activity. In addition, various drug-resistant HIV-1 strains did not display cross resistance to these compounds. Interestingly, time-of-addition experiments indicated that the 4-oxoquinoline derivative remains its anti-HIV-1 activity even after the viral integration stage. Furthermore, the compounds significantly suppressed p24 antigen production in HIV-1 latently infected cells exposed with tumor necrosis factor alpha. These findings suggest that our 4-oxoquinoline derivatives with no 3-carboxylate moiety may become novel lead compounds in the development of anti-HIV-1 drugs.

The Asian project for collaborative derivation of reference intervals: (2) results of non-standardized analytes and transference of reference intervals to the participating laboratories on the basis of cross-comparison of test results.

BACKGROUND: The 2009 Asian multicenter study for derivation of reference intervals (RIs) featured: 1) centralized measurements to exclude reagent-dependent variations; 2) inclusion of non-standardized analytes (hormones, tumor makers, etc.) in the target; and 3) cross-check of test results between the central and local laboratories. Transferability of centrally derived RIs for non-standardized analytes based on the cross-check was examined. METHODS: Forty non-standardized analytes were centrally measured in sera from 3541 reference individuals recruited by 63 laboratories. Forty-four laboratories collaborated in the cross-check study by locally measuring aliquots of sera from 9 to 73 volunteers (average 22.2). Linear relationships were obtained by the major-axis regression. Error in converting RIs using the regression line was expressed by the coefficient of variation of slope b [CV(b)]. CV(b) <10% was set as the cut-off value allowing the conversion. The significance of factors for partitioning RIs was determined similarly as in the first report. RESULTS: Significant sex-, age-, and region-related changes in test results were observed in 17, 15, and 11 of the 40 analytes, respectively. In the cross-comparison study, test results were not harmonized in the majority of immunologically measured analytes, but their average CV(b)s were <10% except for total protein, cystatin C, CA19-9, free thyroxine, and triiodothyronine. After conversion, 74% of centrally derived RIs were transferred to each local laboratory. CONCLUSIONS: Our results point to the feasibility of: 1) harmonizing test results across different laboratories; and 2) sharing centrally derived RIs of non-standardized analytes by means of comparative measurement of a set of commutable specimens.

[Merits of acquiring ISO15189 accreditation].

In Japan, an ISO15189 accreditation system was started in 2005. To date, 47 hospitals have been accredited. In this session, I will present the merits of acquiring accreditation regarding ISO15189 based on our experience. Our hospital has 263 beds. The Clinical Examination Section consists of 12 staff (including 5 part-time workers): 7 in change of sample examination and 5 in charge of physiological examination. The annual number of samples is approximately 150,000. Samples collected on health checkups account for 90%. To improve the quality and service, assessment by third persons has been positively utilized in our hospital. Accreditation regarding ISO9001, ISO14001, ISO27001, privacy mark, hospital function assessment, the functional assessment of "ningen-dock"/health checkup hospitals, labor/hygiene service function assessment, and ISO15189 has been acquired. Patients may not recognize ISO. So, it must be utilized, considering that the acquisition of accreditation is not a goal but a starting point. Furthermore, cost-performance should be improved to achieve utilization-related merits. It is important to not only acquire accreditation but also help clinical staff and patients become aware of some changes/merits. Patients may consult a hospital for the following reasons: confidence in the hospital, and the staffs kind/polite attitudes. Long-term management strategies should be established without pursuing only short-term profits. I will introduce several merits of acquiring accreditation regarding ISO15189. Initially, incidental conditions for bids and appeal points include accreditation regarding ISO15189. Our corporation has participated in some competitive bids regarding health checkup business. In some companies, the bid conditions included ISO acquisition. In our hospital, clinical trials have been positively carried out. For participation in trials, hospitals must pass an institutional examination. However, ISO acquisition facilitates the preparation of documents, leading to successful results. The staffs consciousness has also change. I feel that they try to manage samples from blood collection until reporting laboratory data and interpret laboratory findings strictly. With ISO9001 and ISO14001 activities in our corporation, improvements in consciousness and service have increased the number of patients. Although items other than ISO also improved, ISO played an important role. We could participate in an international collaborative study, "Regional Difference in Laboratory Data and Establishment of Common Reference Ranges", which was mainly designed by Prof. Ichihara, Medical Research Department, Yamaguchi University Graduate School. This also resulted from ISO15189 acquisition. Furthermore, the Japanese Society of "Ningen-Dock" selected Dr. Yoshida, the chairman of the board of directors in our corporation, as a chairman of a meeting that will be held by this society in Asahikawa in 2010 as a result of ISO15189 acquisition by the Clinical Examination Section of our hospital, that is, high-level consciousness. It may be the first case for a middle-/small-scale general hospital to lead a meeting held by such a prestigious society. Thus, I have introduced the merits of ISO15189 acquisition. I hope that our experience will contribute to future ISO activities.