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Chemically-induced liver progenitors (CLiPs) have promising applications in liver regenerative medicine. We aimed to clarify the efficacy of CLiPs for ameliorating fibrosis in a diet-induced nonalcoholic steatohepatitis rat model, since nonalcoholic fatty liver disease is currently recognized as the most common form of chronic liver disease in developed countries.Primary mature hepatocytes were isolated from 7-week-old male Wistar rats. To establish CLiPs, isolated hepatocytes were cultured in differentiation medium composed of Y-27632, A-83-01, and CHIR99021 (YAC medium). As an animal model that reproduces NASH pathophysiology, 6-week-old severe combined immunodeficient (SCID) mice were carefully selected and prepared and fed with choline-deficient, L-amino acid-defined, high-fat diet (HFD). After 12 weeks' HFD feeding, the mice were assigned to continue HFD with or without the administration of rat CLiPs (HFD + CLiPs and HFD-CLiPs, respectively). Rat CLiPs were administered from the spleen. Hepatic fibrosis was semi-quantitatively evaluated according to histology. Liver parenchyma and blood samples were collected for biochemical analyses.Rat CLiPs were positive for CK19 and EpCAM were successfully delivered to the liver. At 8 weeks after CLiPs transplantation, the HFD + CLiPs group showed significantly less positive staining than the HFD-CLiPs group. Alanine aminotransferase significantly improved in the HFD + CLiPs group, as demonstrated by Azan staining and αSMA immunostaining. RT qPCR showed that the liver expression of MMP2 and 9 tended to be higher in the HFD + CLiPs group.The anti-fibrotic effect of CLiPs was demonstrated in the immunodeficient NASH animal model and may have therapeutic applications in humans.

Background Serum microRNAs (miRNAs) have been recognized as potential stable biomarkers for various types of cancer. Considering the clinical applications, there are certain critical requirements, such as minimizing the number of miRNAs, reproducibility in a longitudinal clinical course, and superiority to conventional tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9. This study aimed to identify serum miRNAs that indicate the recurrence of colorectal cancer (CRC), surpassing inter-tumor heterogeneity. Methods We conducted an analysis of 434 serum samples from 91 patients with CRC and 71 healthy subjects. miRNAs were obtained from Toray Co., Ltd, and miRNA profiles were analyzed using a three-step approach. miRNAs that were highly expressed in patients with CRC than in the healthy controls in the screening phase, and those that were highly expressed in the preoperative samples than in the 1-month postoperative samples in the discovery phase, were extracted. In the validation phase, the extracted miRNAs were evaluated in 323 perioperative samples, in chronological order. Results A total of 12 miRNAs (miR-25-3p, miR-451a, miR-1246, miR-1268b, miR-2392, miR-4480, miR-4648, miR-4732-5p, miR-4736, miR-6131, miR-6776-5p, and miR-6851-5p) were significantly concordant with the clinical findings of tumor recurrence, however their ability to function as biomarkers was comparable with CEA. In contrast, the combination of miR-1246, miR-1268b, and miR-4648 demonstrated a higher area under the curve (AUC) than CEA. These three miRNAs were upregulated in primary CRC tissues. Conclusion We identified ideal combinatorial miRNAs to predict CRC recurrence.

Circulating microRNA (miRNA) is a major focus in liquid biopsy studies. The circulating levels of certain miRNAs have been suggested to reflect specific physiological conditions, and several studies have reported their potential use as biomarkers for the detection and prognosis of cancer, as well as for predicting responses to chemotherapy or radiotherapy. Alongside these biomarker studies, research into the effects of specific background factors on circulating miRNA levels is progressing. Indeed, several studies have shown that a number of factors, including blood sample collection and processing methods, as well as subject-specific factors such as age, sex, and other physiological conditions, can affect the normal levels of circulating miRNAs. Unfortunately, the evidence supporting these effects is not yet strong enough to support a definite conclusion and further research is warranted. Here, we summarize the findings of several studies that have addressed these concerns and identify important topics that should be considered when analyzing circulating miRNA levels in liquid biopsy studies.

Although radical prostatectomy is associated with good long-term oncological outcomes, approximately 30% of patients present biochemical recurrence, whereupon salvage treatments are required. Identification of novel molecular biomarkers to predict cancer behavior is clinically important. Here, we developed a novel microRNA (miRNA)-based prognostic model for patients who underwent radical prostatectomy.We retrospectively investigated the clinical records of 295 patients who underwent radical prostatectomy between 2009 and 2017. We randomly assigned these cases into training or validation sets. The prognostic model was constructed using Fisher linear discriminant analysis in the training set, and we evaluated its performance in the validation set.Overall, 72 patients had biochemical recurrence. A prediction model was constructed using a combination of three miRNAs (miR-3147, miR-4513, and miR-4728-5p) and two pathological factors (pathological T stage and Gleason score). In the validation set, the predictive performance of the model was confirmed to be accurate (area under the receiver operating characteristic curve: 0.80; sensitivity: 0.78; specificity: 0.76). Additionally, Kaplan-Meier analysis revealed that the patients with a low prediction index had significantly longer recurrence-free survival than those with a high index (p 0.001).Circulating miRNA profiles can provide information to predict recurrence after prostatectomy. Our model may be helpful for physicians to decide follow-up strategies for patients.

Extracellular vesicles (EVs) have emerged as immunomodulatory regulators during tumor progression. These small vesicles encapsulate a variety of molecules, including DNA, RNA, and proteins. When EVs come in contact with recipient cells, the EVs transmit various physiological characteristics; for example, proteins on the surface of EVs act as ligands. Immune checkpoint blockade targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has shown promise in a subset of cancer patients. PD-L1 on EVs acts as a key immunomodulator. Suppression of EV secretion enhances the efficacy of immunotherapy using immune checkpoint blockade antibodies. In addition to immune checkpoint blockade therapy, chimeric antigen receptor T (CAR-T) cell therapy has also been used to successfully eliminate cancer cells. Interestingly, CAR-T-cell-derived EVs express CAR on their surface. Compared with CAR-T cells, CAR-expressing EVs do not express PD1, so their antitumor effect cannot be weakened. In this review, we describe the current understanding of EVs in cancer immunity and summarize their crucial roles in immunomodulation.

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC. Significance: Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

Restoration of lymphatic drainage across the bronchial anastomosis after lung transplantation requires several weeks. As donor antigen and antigen presenting cell trafficking via lymphatics into graft-draining lymph nodes is an important component of the alloresponse, alternative pathways must exist that account for rapid rejection after pulmonary transplantation. Here, we describe a novel allorecognition pathway mediated through donor extracellular vesicle (EV) trafficking to mediastinal lymph nodes via the pleural space. Pleural fluid collected early after lung transplantation in rats and humans contains donor-specific EVs. In a fully MHC mismatched rat model of lung transplantation, we demonstrate EVs carrying donor antigen preferentially accumulate in mediastinal lymph nodes and colocalize with MHC II expressing cells within 4 h of engraftment. Injection of allogeneic EVs into pleural space of syngeneic lung transplant recipients confirmed their selective trafficking to mediastinal lymph nodes and resulted in activation of T cells in mediastinal, but not peripheral lymph nodes. Thus, we have uncovered an alternative pathway of donor antigen trafficking where pulmonary EVs released into the pleural space traffic to locoregional lymph nodes via pleural lymphatics. This pathway obviates the need for restoration of lymphatics across the bronchial anastomosis for trafficking of donor antigen to draining lymph nodes.

We investigated umbilical cord serum microRNA (miRNA) profiles to identify biomarkers of a risk for obesity later in life. Participating children were divided into high- and low-risk groups of obesity based on the timing of adiposity rebound and the body mass index (BMI) at 5 years and randomly selected from each group for this study. 3D-Gene® Human miRNA Oligo Chip was performed using cord serum in five children of both groups. The most relevant miRNAs were confirmed in 33 children of the groups using the TaqMan® microRNA assay. We detected five cord serum miRNAs differentially expressed in children at high risk of obesity compared with the levels in children at low risk, namely, miR-516-3p and miR-130a-3p with increased levels and miR-1260b, miR-4709-3p, and miR194-3p with decreased levels. This study provides the first identification of altered umbilical cord serum miRNAs in childhood obesity.

Background Small hepatocyte-like progenitor cells (SHPCs) are hepatocytic progenitor cells that transiently form clusters in rat livers treated with retrorsine (Ret) that underwent 70% partial hepatectomy (PH). We previously reported that transplantation of Thy1+ cells obtained from d-galactosamine-treated livers promotes SHPC expansion, thereby accelerating liver regeneration. Extracellular vesicles (EVs) secreted by Thy1+ cells induce sinusoidal endothelial cells (SECs) and Kupffer cells (KCs) to secrete IL17B and IL25, respectively, thereby activating SHPCs through IL17 receptor B (RB) signaling. This study aimed to identify the inducers of IL17RB signaling and growth factors for SHPC proliferation in EVs secreted by Thy1+ cells (Thy1-EVs). Methods Thy1+ cells isolated from the livers of rats treated with d-galactosamine were cultured. Although some liver stem/progenitor cells (LSPCs) proliferated to form colonies, others remained as mesenchymal cells (MCs). Thy1-MCs or Thy1-LSPCs were transplanted into Ret/PH-treated livers to examine their effects on SHPCs. EVs were isolated from the conditioned medium (CM) of Thy1-MCs and Thy1-LSPCs. Small hepatocytes (SHs) isolated from adult rat livers were used to identify factors regulating cell growth in Thy1-EVs. Results The size of SHPC clusters transplanted with Thy1-MCs was significantly larger than that of SHPC clusters transplanted with Thy1-LSPCs (p = 0.02). A comprehensive analysis of Thy1-MC-EVs revealed that miR-199a-5p, cytokine-induced neutrophil chemoattractant-2 (CINC-2), and monocyte chemotactic protein 1 (MCP-1) were candidates for promoting SHPC growth. Additionally, miR-199a-5p mimics promoted the growth of SHs (p = 0.02), whereas CINC-2 and MCP-1 did not. SECs treated with CINC-2 induced Il17b expression. KCs treated with Thy1-EVs induced the expression of CINC-2, Il25, and miR-199a-5p. CM derived from SECs treated with CINC-2 accelerated the growth of SHs (p = 0.03). Similarly, CM derived from KCs treated with Thy1-EVs and miR-199a-5p mimics accelerated the growth of SHs (p = 0.007). In addition, although miR-199a-overexpressing EVs could not enhance SHPC proliferation, transplantation of miR-199a-overexpressing Thy1-MCs could promote the expansion of SHPC clusters. Conclusion Thy1-MC transplantation may accelerate liver regeneration owing to SHPC expansion, which is induced by CINC-2/IL17RB signaling and miR-199a-5p via SEC and KC activation. Graphical Abstract

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.

Table S1. Synthetic DNA oligomers, PCR primers

Supplementary Fig. S1. Nedd9 and Aurkb seed match sequences in mucAB and umuDC.Supplementary Fig. S2. Selection of cells expressing Tet-repressor protein.Supplementary Fig. S3. Microarray analysis of deregulated oncogenes.Supplementary Fig. S4. Suppression of cellular transformation with anti-mucAB shRNA.Supplementary Fig. S5. Confirmation of interaction of miR-145 with anti-miR-145.Supplementary Fig. S6. Microcolony formation over time.Supplementary Fig. S7. Comparison of tumor incidence rates between males and females, and between presence and absence of Zn.

Supplementary Data from Single-Cell Transcriptome Profiling Reveals Intratumoral Heterogeneity and Molecular Features of Ductal Carcinoma In Situ

Purpose:The high false-positive rate of prostate-specific antigen (PSA) may lead to unnecessary prostate biopsies. Therefore, the United States Preventive Services Task Force recommends that decisions regarding PSA-based screening of prostate cancer should be made with caution in men ages 55–69 years, and that men ≥70 years should not undergo PSA screening. Here, we investigated the potential of serum miRNAs as an accurate diagnostic method in patients with suspected prostate cancer.Experimental Design:Serum samples of 809 patients with prostate cancer, 241 negative prostate biopsies, and 500 patients with other cancer types were obtained from the National Cancer Center, Japan. Forty-one healthy control samples were obtained from two other hospitals in Japan. Comprehensive microarray analysis was performed for all samples. Samples were divided into three sets. Candidate miRNAs for prostate cancer detection were identified in the discovery set (n = 123). A diagnostic model was constructed using combinations of candidate miRNAs in the training set (n = 484). The performance of the diagnostic model was evaluated in the validation set (n = 484).Results:In the discovery set, 18 candidate miRNAs were identified. A robust diagnostic model was constructed using the combination of two miRNAs (miR-17-3p and miR-1185-2-3p) in the training set. High diagnostic performance with a sensitivity of 90% and a specificity of 90% was achieved in the validation set regardless of the Gleason score and clinical tumor–node–metastasis stage.Conclusions:The model developed in this study may help improve the diagnosis of prostate cancer and reduce the number of unnecessary prostate biopsies.

Supplementary materials and methods

Purpose: The outcome of patients with advanced hepatocellular carcinoma (HCC) has remained unsatisfactory. Patients with HCC suffer from chronic hepatitis or liver cirrhosis, and their reserve liver function is often limited.Experimental Design: To develop new therapeutic agents that act specifically on HCC but interfere only minimally with residual liver function, we searched for genes that were upregulated in 20 cases of HCC [namely, discovery sets 1 (n = 10) and 2 (n = 10)] in comparison with corresponding nontumorous liver and a panel representing normal organs using high-density microarrays capable of detecting all exons in the human genome.Results: Eleven transcripts whose expression was significantly increased in HCC were subjected to siRNA-based secondary screening of genes required for HCC cell proliferation as well as quantitative reverse transcription-PCR analysis [validation sets 1 (n = 20) and 2 (n = 44)] and immunohistochemistry (n = 19). We finally extracted four genes, AKR1B10, HCAP-G, RRM2, and TPX2, as candidate therapeutic targets for HCC. siRNA-mediated knockdown of these candidate genes inhibited the proliferation of HCC cells and the growth of HCC xenografts transplanted into immunodeficient mice.Conclusions: The four genes we identified were highly expressed in HCC, and HCC cells are highly dependent on these genes for proliferation. Although many important genes must have been overlooked, the selected genes were biologically relevant. The combination of genome-wide expression and functional screening described here is a rapid and comprehensive approach that could be applied in the identification of therapeutic targets in any type of human malignancy. Clin Cancer Res; 16(9); 2518–28. ©2010 AACR.

Figure S1-3, Table S1-4

Figure S1. Schematic representation of workflow for single-cell isolation and sc-qPCR. Figure S2. Violin plot of each individual gene expression along drug resistance acquisition. Figure S3. Correlation of several genes expressed at the single-cell level, separated by cell type as represented by the color of each dot. Figure S4. Lef1, Vim and Cav1 protein expression in several Luminal breast cancer cell lines. Figure S5. Larger visual fields showing immunostaining in tissue samples. Figure S6. Clinical information regarding the role of Lef1 in stained tissue samples. Figure S7. Comparison of Lef1 protein levels in MCF7 and derivatives.

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC.Significance:Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

Purpose:Sentinel lymph node biopsy (SLNB) is the gold-standard procedure for evaluating axillary lymph node (ALN) status in patients with breast cancer. However, the morbidity of SLNB is not negligible, and the procedure is invasive for patients without ALN metastasis. Here, we developed a diagnostic model for evaluating ALN status using a combination of serum miRNAs and clinicopathologic factors as a novel less-invasive biomarker.Experimental Design: Preoperative serum samples were collected from patients who underwent surgery for primary breast cancer or breast benign diseases between 2008 and 2014. A total of 958 serum samples (921 cases of primary breast cancer, including 630 cases in the no ALN metastasis group and 291 cases in the ALN metastasis group, and 37 patients with benign breast diseases) were analyzed by miRNA microarray. Samples were randomly divided into training and test sets. Logistic LASSO regression analysis was used to construct diagnostic models in the training set, which were validated in the test set.Results:An optimal diagnostic model was identified using a combination of two miRNAs (miR-629-3p and miR-4710) and three clinicopathologic factors (T stage, lymphovascular invasion, and ultrasound findings), which showed a sensitivity of 0.88 (0.84–0.92), a specificity of 0.69 (0.61–0.76), an accuracy of 0.818, and an area under the receiver operating characteristic curve of 0.86 in the test set.Conclusions:Serum miRNA profiles may be useful for the diagnosis of ALN metastasis before surgery in a less-invasive manner than SLNB.

Table S1. List of TaqMan probes used for the 95-primer set.

Supplementary Data from Combined Functional Genome Survey of Therapeutic Targets for Hepatocellular Carcinoma

Table S2. Characteristic of clinical samples

Table S1. Definition criteria of categorical variables for logistic-LASSO regression analysis Table S2. Clinicopathological characteristics of patients with benign breast diseases Table S3. Clinicopathological characteristics of patients stratified into the training set and test set Table S4. Comparison of T staging between pre- and post-operative diagnosis Table S5. Comparison of lymphovascular invasion (LVI) between pre- and post-operative diagnosis Table S6. Clinicopathological characteristics of N-positive patients misdiagnosed as N-negative in the test set Table S7. Sensitivity and specificity analysis for the diagnostic index in the test set Table S8. Biomarker candidate miRNAs for N positivity Table S9. Comparison of post-operative lymphovascular invasion (LVI) between original and re-evaluated diagnosis in the training set Table S10. Characteristics of ultrasound findings of axillary lymph node status Table S11. Utility of ultrasonography for the detection of axillary lymph node metastasis Figure S1. Ultrasound findings of suspicious metastatic and negative axillary lymph nodes. Figure S2. Serum levels of miR-629-3p and miR-4710 in the training set. P-values were calculated by one-way ANOVA with Tukey's post-hoc analysis. Figure S3. ROC curves for histological grade and Ki-67 labeling index in the test set (postoperative pathology). Numbers in parentheses are 95% confidence intervals. Text. Materials and Methods

Supplementary Figure from Single-Cell Transcriptome Profiling Reveals Intratumoral Heterogeneity and Molecular Features of Ductal Carcinoma In Situ

Supplementary text

Drug resistance is a major obstacle in the treatment of breast cancer. Surviving cells lead to tumor recurrence and metastasis, which remains the main cause of cancer-related mortality. Breast cancer is also highly heterogeneous, which hinders the identification of individual cells with the capacity to survive anticancer treatment. To address this, we performed extensive single-cell gene-expression profiling of the luminal-type breast cancer cell line MCF7 and its derivatives, including docetaxel-resistant cells. Upregulation of epithelial-to-mesenchymal transition and stemness-related genes and downregulation of cell-cycle–related genes, which were mainly regulated by LEF1, were observed in the drug-resistant cells. Interestingly, a small number of cells in the parental population exhibited a gene-expression profile similar to that of the drug-resistant cells, indicating that the untreated parental cells already contained a rare subpopulation of stem-like cells with an inherent predisposition toward docetaxel resistance. Our data suggest that during chemotherapy, this population may be positively selected, leading to treatment failure.Significance:This study highlights the role of breast cancer intratumor heterogeneity in drug resistance at a single-cell level.

Supplementary Fig. S1. Spheroid cells derived from human ovarian cancer show characteristics of CSCs. Supplementary Fig. S2. High levels of ALDH activity are associated with CSC-related characteristics. Supplementary Fig. S3. ALDH activity is required for SOX2 expression and proliferation of ovarian CSCs. Supplementary Fig. S4. ALDH1A1 mediates SOX2 expression and proliferation of ovarian CSCs. Supplementary Fig. S5. Feedback regulation by ALDH1A1 and SOX2 for the proliferation of ovarian CSCs.

Supplementary Table S1. Information on clinical data of SOCs and sphere formation. Supplementary Table S2. Xenograft tumor formation assay of the ovarian spheroids (#4). Supplementary Table S3. Limited dilution assay on the FACS-sorted spheroid cells. Supplementary Table S4. Clinicopathological features of high-grade serous ovarian carcinoma patients. Supplementary Table S5. Statistical analyses of ALDH1A1 expression in advanced-stage high-grade serous ovarian carcinoma. Supplementary Table S6. Limited dilution assays for the sh-ALDH1A1-infected cells. Supplementary Table S7. Limited dilution assays for the sh-SOX2-infected cells.

Legends for supplementary Figures 1-5

The establishment of cancer stem-like cell (CSC) culture systems may be instrumental in devising strategies to fight refractory cancers. Inhibition of the Rho kinase ROCK has been shown to favorably affect CSC spheroid cultures. In this study, we show how ROCK inhibition in human serous ovarian cancer (SOC) cells can help establish a CSC system, which illuminates cancer pathophysiology and its treatment in this setting. In the presence of a ROCK kinase inhibitor, spheroid cultures of SOC cells expressed characteristic CSC markers including ALDH1A1, CD133, and SOX2, along with differentiation and tumorigenic capabilities in mouse xenograft models of human SOC. High expression levels of ALDH, but not CD133, correlated with spheroid formation CSC marker expression and tumor forming capability. In clinical specimens of SOC, high levels of ALDH1A1 correlated with advanced stage and poor prognosis. Pharmacologic or genetic blockade of ALDH blocked cell proliferation and reduced expression of SOX2, the genetic ablation of which abolished spheroid formation, whereas SOX2 overexpression inhibited ALDH1A1 expression and blocked spheroid proliferation. Taken together, our findings illustrated a new method to culture human ovarian CSC, and they defined a reciprocal regulatory relationship between ALDH1A1 and SOX2, which impacts ovarian CSC proliferation and malignant progression. Cancer Res; 76(1); 150–60. ©2015 AACR.

Lymphovascular invasion (LVI) is a fundamental step toward the spread of cancer. Extracellular vesicles (EVs) promote cellular communication by shuttling cargo, such as microRNAs (miRNAs). However, whether EV-associated miRNAs serve as biomarkers for LVI remains unclear. This study aimed to identify EV-associated miRNAs related to LVI and validate the miRNA levels from patients with early-stage lung adenocarcinoma (LADC). Blood samples were collected from patients undergoing pulmonary resection for stage I LADC before surgery. The patients were classified into three groups according to the presence of LVI and postoperative recurrence. Serum-derived EVs in the derivation cohort were used for small RNA sequencing, while the selected LVI miRNA candidates were validated via real-time quantitative polymerase chain reaction using 44 patient and 16 healthy donor samples as the validation cohorts. Five miRNAs (miR-99b-3p, miR-26a-5p, miR-93-5p, miR-30d-5p, and miR-365b-3p) were assessed, and miR-30d-5p (p = 0.036) levels were significantly downregulated in the LVI-positive group. miR-30d-5p levels in healthy donors were lower than those in LADC patients. Patients with high miR-30d-5p levels had favorable survival compared to those with low miR-30d-5p levels. miR-30d-5p level in EVs may serve as a promising biomarker for detecting LVI in patients with early-stage LADC.

Radical cystectomy is the gold-standard treatment for muscle-invasive bladder cancer and aggressive non-muscle-invasive bladder cancer. To enhance clinical decision-making regarding patients with bladder cancer who underwent radical cystectomy, a recurrence prediction biomarker with high accuracy is urgently needed. In this study, we developed a model for the prediction of bladder cancer recurrence after radical cystectomy by combining serum microRNA and a pathological factor.We retrospectively analyzed the clinical records of 81 patients with bladder cancer who underwent radical cystectomy between 2008 and 2016. The dataset was divided into two, and Fisher linear discriminant analysis was used to construct a prognostic model for future recurrence in the training set (n = 41). The performance of the model was evaluated in the validation set (n = 40).Thirty patients had recurrence after having undergone radical cystectomy. A prognostic model for recurrence was constructed by combining a pathological factor (i.e. positive pathological lymph node status) and three microRNAs (miR-23a-3p, miR-3679-3p, and miR-3195). The model showed a sensitivity of 0.87, a specificity of 0.80, and an area under the receiver operating characteristic curve of 0.88 (0.77-0.98) in the validation set. Furthermore, Kaplan-Meier analysis revealed that patients with a low prediction index have significantly longer overall survival than patients with a high prediction index (P = 0.041).A combination of serum microRNA profiles and lymph node statuses is useful for the prediction of oncological outcomes after radical cystectomy in patients with bladder cancer.

Background Small hepatocyte-like progenitor cells (SHPCs) are hepatocytic progenitor cells that transiently form clusters in rat livers treated with retrorsine and with 70% partial hepatectomy (PH). We previously reported that transplantation of Thy1+ cells derived from d-galactosamine-treated livers promotes SHPC expansion, resulting in the acceleration of liver regeneration. Extracellular vesicles (EVs) produced by Thy1+ cells act on sinusoidal endothelial cells (SECs) and Kupffer cells to secrete IL17B and IL25, respectively, resulting in SHPC activation through IL17 receptor B (RB) signaling. Our aim is to identify factors in Thy1-EVs that activate IL17RB signaling. Methods Thy1+ cells isolated from rats with d-galactosamine-induced liver injury were cultured for one week. Although some liver stem/progenitor cells proliferated into colonies, others maintained as mesenchymal cells (MCs). Thy1-MCs or Thy1-liver stem/progenitor cells were transplanted into retrorsine/PH-treated livers to examine their effects on SHPCs. SHs isolated from adult rat livers were used to validate factors regulating growth induction. Results The number and size of SHPCs remarkably increased in livers transplanted with Thy1-MCs. Comprehensive analysis of Thy1-MC-EVs revealed that miR-199a-5p, CINC-2, and MCP-1 are candidates for stimulating SHPC growth. Administration of the miR-199a-5p mimic, and not CINC-2, promoted SH growth. SECs treated with CINC-2 induced IL17b expression and their conditioned medium promoted SH growth. Conclusion Thy1-MC transplantation may accelerate liver regeneration due to SHPCs expansion, which is stimulated by CINC-2/IL17RB signaling and miR-199a-5p.

SUMMARYOver the last several years, a method has emerged which endows adult hepatocytes within vitroproliferative capacity, producing chemically-induced liver progenitors (CLiPs). However, a recent study questioned the origin of these cells, suggesting that resident liver progenitor cells, but not hepatocytes, proliferate. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacityin vitro. Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate chronically injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. Moreover, this study offers bilCLiPs as a potential cell source for liver regenerative medicine.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian carcinoma with poor prognosis. However, no effective biomarkers have been established for predicting unfavorable events, including recurrence and poor prognoses. Serum microRNAs (miRNAs) have been increasingly reported to be useful in predicting a patient's condition and have been recognized as a potentially less-invasive source for liquid biopsy in cancer. Therefore, this study aimed to evaluate serum miRNA profiles from patients with OCCC and to establish biomarker for predicting the prognoses.The GSE106817, which included preoperative serum miRNA profiles of patients with ovarian tumors, was used, and clinical information was investigated. In all, 66 patients with OCCC were included, excluding those with other histological subtypes or insufficient prognostic information. Moreover, miRNA profiles of OCCC tissues were also examined.The median follow-up period was 64.3 (8.0-153.3) months. Based on multivariable Cox regression analyses and the expression of miRNAs in OCCC tissues, miR-150-3p, miR-3195, and miR-7704 were selected as miRNA candidates associated with both progression-free survival (PFS) and overall survival (OS). Then, the prognostic index was calculated based on expression values of 3 serum miRNAs. Kaplan-Meier survival analysis indicated that the prognostic index was significantly predictive of PFS and OS (p=0.004 and p=0.012, respectively).Preoperative serum miRNA profiles of miR-150-3p, miR-3195, and miR-7704 can be used to potentially predict the prognosis of patients with OCCC.

Vascular invasion (VI) is a fundamental step toward the spread of cancer. Extracellular vesicles (EVs) promote cellular communication by shuttling cargo, such as microRNAs (miRNAs). However, whether EV-associated miRNAs serve as biomarkers for VI remains unclear. This study aimed to identify EV-associated miRNAs related to VI and validate the miRNA levels from patients with early-stage lung adenocarcinoma (LADC). Blood samples were collected from patients undergoing pulmonary resection for stage I LADC before surgery. The patients were classified into three groups according to the presence of VI and postoperative recurrence. Serum-derived EVs in the derivation cohort were used for small RNA sequencing, while the selected VI miRNA candidates were validated via real-time quantitative polymerase chain reaction using 44 patient and 16 healthy donor samples as the validation cohorts. Five miRNAs (miR-99b-3p, miR-26a-5p, miR-93-5p, miR-30d-5p, and miR-365b-3p) were assessed, and miR-30d-5p (p = 0.036) levels were significantly downregulated in the VI-positive group. MiR-30d-5p levels in healthy donors were lower than those in LADC patients. Patients with high miR-30d-5p levels had favorable survival compared to those with low miR-30d-5p levels. MiR-30d-5p level in EVs may serve as a promising biomarker for detecting VI in patients with early-stage LADC.

A major obstacle to the implementation of early palliative care (EPC) is the lack of objective criteria for referral to EPC. Circulating microRNAs (miRNAs) have been recognized as promising biomarkers. The present study investigated objective definitions for referral to EPC using microRNA. A total of 178 serum samples were obtained from patients with lung, gastrointestinal, colorectal, bile duct, pancreas and bladder cancers who were treatment-naïve and received chemotherapy between January 2011 and December 2013 at National Cancer Center Hospital East. We investigated expression levels of miRNAs using microarrays. The primary outcome was prediction of admission to a palliative care unit ≤6 months after first visit. Diagnostic models using clinical characteristics, miRNAs and combinations of both were constructed. The miRNA models were constructed using 6 miRNA levels. The best areas under the receiver operating characteristic curve (AUCs) of the clinical model was 0.741, while the average AUCs of miRNA-based models and combination models were 0.769 and 0.806, respectively. Combination models showed higher AUCs than the clinical model (

High‐grade serous ovarian carcinoma is a leading cause of death in female patients worldwide. MicroRNAs (miRNAs) are stable noncoding RNAs in the peripheral blood that reflect a patient’s condition, and therefore, they have received substantial attention as noninvasive biomarkers in various diseases. We previously reported the usefulness of serum miRNAs as diagnostic biomarkers. Here, we investigated the prognostic impact of the serum miRNA profile. We used the GSE106817 dataset, which included preoperative miRNA profiles of patients with ovarian malignancies. Excluding patients with other malignancy or insufficient prognostic information, we included 175 patients with high‐grade serous ovarian carcinoma. All patients except four underwent surgery and received chemotherapy as initial treatment. The median follow‐up period was 54.6 months (range, 3.5‐144.1 months). Univariate Cox regression analysis revealed that higher levels of miR‐187‐5p and miR‐6870‐5p were associated with both poorer progression‐free survival (PFS) and overall survival (OS), and miR‐1908‐5p, miR‐6727‐5p, and miR‐6850‐5p were poor prognostic indicators of PFS. The OS and PFS prognostic indices were then calculated using the expression values of three prognostic miRNAs. Multivariate Cox regression analysis showed that both indices were significantly independent poor prognostic factors (hazard ratio for OS and PFS, 2.343 [P = .015] and 2.357 [P = .005], respectively). In conclusion, circulating miRNA profiles can potentially provide information to predict the prognosis of patients with high‐grade serous ovarian carcinoma. Therefore, there is a strong demand for early clinical application of circulating miRNAs as noninvasive biomarkers., We showed that several circulating microRNAs were useful as predictive biomarkers in patients with high‐grade serous ovarian carcinoma. This study and our previous report indicated that circulating microRNA profiles can be useful clinical tools for a highly accurate diagnosis and prognostic prediction using a single blood test.

Background Noninvasive detection of early stage cancers with accurate prediction of tumor tissue-of-origin could improve patient prognosis. Because miRNA profiles differ between organs, circulating miRNomics represent a promising method for early detection of cancers, but this has not been shown conclusively. Methods A serum miRNA profile (miRNomes)–based classifier was evaluated for its ability to discriminate cancer types using advanced machine learning. The training set comprised 7931 serum samples from patients with 13 types of solid cancers and 5013 noncancer samples. The validation set consisted of 1990 cancer and 1256 noncancer samples. The contribution of each miRNA to the cancer-type classification was evaluated, and those with a high contribution were identified. Results Cancer type was predicted with an accuracy of 0.88 (95% confidence interval [CI] = 0.87 to 0.90) in all stages and an accuracy of 0.90 (95% CI = 0.88 to 0.91) in resectable stages (stages 0-II). The F1 score for the discrimination of the 13 cancer types was 0.93. Optimal classification performance was achieved with at least 100 miRNAs that contributed the strongest to accurate prediction of cancer type. Assessment of tissue expression patterns of these miRNAs suggested that miRNAs secreted from the tumor environment could be used to establish cancer type–specific serum miRNomes. Conclusions This study demonstrates that large-scale serum miRNomics in combination with machine learning could lead to the development of a blood-based cancer classification system. Further investigations of the regulating mechanisms of the miRNAs that contributed strongly to accurate prediction of cancer type could pave the way for the clinical use of circulating miRNA diagnostics.

Cardiac diseases such as myocardial infarction and heart failure have been the leading cause of death worldwide for more than 20 years, and new treatments continue to be investigated. Heart transplantation, a curative treatment for severe cardiac dysfunction, is available to only a small number of patients due to the rarity of donors and high costs. Cardiac regenerative medicine using embryonic stem cells and induced pluripotent stem cells is expected to be a new alternative to heart transplantation, but it has problems such as induction of immune response, tumor formation, and low survival rate of transplanted cells. On the other hand, there has been a focus on cell-free therapy using extracellular vesicles (EVs) due to their high biocompatibility and target specificity. Exosomes, one type of EV, play a role in the molecular transport system in vivo and can be considered a drug delivery system (DDS) innate to all living things. Exosomes contain nucleic acids and proteins, which are transported from secretory cells to recipient cells. Molecules in exosomes are encapsulated in a lipid bilayer, which allows them to exist stably in body fluids without being affected by nuclease degradation enzymes. Therefore, the therapeutic use of exosomes as DDSs has been widely explored and is being used in clinical trials and other clinical settings. This review summarizes the current topics of EVs as DDSs in cardiac disease.

Cancer stem cells (CSCs) are a small cell subpopulation in many cancer types and are involved in various processes of tumor progression, such as initiation, metastasis and recurrence. The distinguished features of CSCs include a variety of biological properties, including self-renewal, multidifferentiation, stemness marker expression, and resistance to chemotherapy and radiotherapy. Despite their great potential of clinical importance, the CSC signaling pathways are not well understood at the molecular level. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs that play an important role in the regulation of several cellular, physiological, and developmental processes. Aberrant miRNA expression is associated with many human diseases, including cancer. miRNAs have been implicated in the regulation of CSC properties; therefore, a better understanding of miRNA-induced modulation of CSC gene expression could aid in the identification of promising biomarkers and therapeutic targets. In the present review, we summarize the major findings of the impacts of miRNAs on CSC signaling networks; we then discuss the recent advances that have improved our understanding of CSC regulation by miRNA-mediated signaling networks and that may lead to the development of miRNA therapeutics specifically targeting CSCs.