In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.

Yes-associated protein (YAP) transcriptional coactivator is negatively regulated by the Hippo pathway and functions in controlling the size of multiple organs, such as liver during development. However, it is not clear whether YAP signaling participates in the process of the formation of glia scars after spinal cord injury (SCI). In this study, we found that YAP was upregulated and activated in astrocytes of C57BL/6 male mice after SCI in a Hippo pathway-dependent manner. Conditional knockout (KO) of yap in astrocytes significantly inhibited astrocytic proliferation, impaired the formation of glial scars, inhibited the axonal regeneration, and impaired the behavioral recovery of C57BL/6 male mice after SCI. Mechanistically, the bFGF was upregulated after SCI and induced the activation of YAP through RhoA pathways, thereby promoting the formation of glial scars. Additionally, YAP promoted bFGF-induced proliferation by negatively controlling nuclear distribution of p27 Kip1 mediated by CRM1. Finally, bFGF or XMU-MP-1 (an inhibitor of Hippo kinase MST1/2 to activate YAP) injection indeed activated YAP signaling and promoted the formation of glial scars and the functional recovery of mice after SCI. These findings suggest that YAP promotes the formation of glial scars and neural regeneration of mice after SCI, and that the bFGF-RhoA-YAP-p27 Kip1 pathway positively regulates astrocytic proliferation after SCI.

ABSTRACT

BackgroundEpithelial–mesenchymal transition (EMT) is implicated in the metastatic process and presents a challenge to epithelial cell adhesion molecule-based detection of circulating tumor cells (CTCs), which have been demonstrated to be a prognostic indicator in metastatic breast cancer. Although evidence has indicated that heterogeneity of CTCs based on EMT markers is associated with disease progression, no standard recommendations have been established for clinical practice. This study aimed to evaluate the prognostic significance of dynamic CTC detection based on EMT for metastatic breast cancer patients.MethodsWe enrolled 108 human epidermal growth factor receptor 2-negative metastatic breast cancer patients from the prospective phase III CAMELLIA study and applied the CanPatrol CTC enrichment technique to identify CTC phenotypes (including epithelial CTCs, biphenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs) in peripheral blood samples. Receiver operating characteristic curve analyses of total CTC count and the proportion of mesenchymal CTCs for predicting the 1-year progression-free survival (PFS) rate were conducted to determine the optimal cut-off values, and Kaplan–Meier analysis and Cox proportional hazards regression analysis were performed to investigate the prognostic value of the cut-off values of both total CTC count and the proportion of mesenchymal CTCs in combination.ResultsFor predicting the 1-year PFS rate, the optimal cut-off value of total CTC count was 9.5 (Area under the curve [AUC] = 0.538, 95% confidence interval [CI] = 0.418–0.657), and that of the proportion of mesenchymal CTCs was 10.7% (AUC = 0.581, 95% CI = 0.463–0.699). We used the two cut-off values in combination to forecast PFS in which the total CTC count was equaled to or exceeded 10/5 mL with the proportion of mesenchymal CTCs surpassed 10.7%. Patients who met the combined criteria had significantly shorter median PFS than did those who did not meet the criteria (6.2 vs. 9.9 months, P =0.010). A nomogram was constructed based on the criteria and significant clinicopathological characteristics with a C-index of 0.613 (P = 0.010).ConclusionsThe criteria, which combine the total CTC count and the proportion of mesenchymal CTCs, may be used to monitor therapeutic resistance and predict prognosis in patients with metastatic breast cancer.Trial registration ClinicalTrials.gov. NCT01917279. Registered on 19 July 2013, https://clinicaltrials.gov/ct2/show/NCT01917279?term=NCT01917279&rank=1.

BackgroundCirculating tumor cells (CTCs), an advantageous target of liquid biopsy, is an important biomarker for the prognosis and monitoring of cancer. Currently, detection techniques for CTCs are mainly based on the physical and/or epithelial characteristics of tumor cells. However, biofunctional activity markers that can indicate the high metastatic capacity of CTCs are lacking.MethodsFunctional microarray, quantitative real-time polymerase chain reaction, and Western blot were used on five prostate cancer cell lines with different metastatic capacities to identify the metastasis-related metabolic genes. The identified genes were detected in the CTCs of 64 clinical samples using the RNA in situ hybridization. A multi-criteria weighted model was used to determine the optimal metabolic markers for the CTCs test. Based on five fluorescent signals targeting DAPI, CD45, metabolic, epithelial (EpCAM/CKs), and mesenchymal (Vimentin/Twist) markers, the filtration-enriched CTCs were classified as GM+CTCs/GM−CTCs (metabolic types) or E-CTCs/H-CTCs/M-CTCs (EMT types). Correlation analysis and ROC curve were conducted on 54 prostate cancer samples to evaluate the clinical significance of CTCs subtypes.ResultsEight metastasis-related metabolic genes were identified, including HK2, PDP2, G6PD, PGK1, PHKA1, PYGL, PDK1, and PKM2. Among them, PGK1 and G6PD were determined as optimal glucose metabolic (GM) markers for CTCs. GM+CTCs (marked by PGK1/G6PD) were detectable in 64.8% (35/54) of prostate cancer patients, accounting for 46.5% (134/288) of total CTCs. An increased GM+CTCs level was associated with advanced tumor stage and metastasis (P <  0.05). In the discrimination of cancer metastasis from non-metastasis, GM+CTCs presented a higher AUC of the ROC curve (0.780) compared with the EMT CTCs subtypes (E-CTCs 0.729, H-CTCs 0.741, and M-CTCs 0.648). A triple tPSA–Gleason–GM+CTCs marker increased the AUC to 0.904, which was better than that of the tPSA–Gleason–H-CTCs marker (0.874).ConclusionsThe metabolic marker (PGK1/G6PD) is determined as the indicator for the biofunctional activity analysis of CTCs, compared with the existing morphological (EMT) classification on CTCs. The metabolic characterization of CTCs demonstrates that hypermetabolic GM+CTCs are promising biomarkers for prostate cancer metastasis.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0789-0) contains supplementary material, which is available to authorized users.

Objective: Nasopharyngeal carcinoma is one of the leading malignancies with obscure etiology. Circulating tumor cells have been showed to intimately correlate with characteristics in different kinds of cancer. But links between circulating tumor cells and nasopharyngeal carcinoma were still lacking. Therefore, we explored circulating tumor cells' distribution in nasopharyngeal carcinoma and their possible associations with nasopharyngeal carcinoma. Methods: Firstly, we found that the positive ratio of circulating tumor cells is extremely high in four stages of nasopharyngeal carcinoma. Meanwhile, positive ratios of mesenchymal circulating tumor cells were higher in early stages of nasopharyngeal carcinoma. Apart from epithelial circulating tumor cells, total, hybrid and mesenchymal circulating tumor cells were correlated with nasopharyngeal carcinoma clinical stage. Results: Our results showed that hybrid and mesenchymal circulating tumor cells were associated with nasopharyngeal carcinoma metastasis (both distant and lymph node) and smoking. Meanwhile, hybrid circulating tumor cells expressed the highest Epstein-Barr virus proteins and deoxyribonucleic acid in three types of circulating tumor cells. Moreover, we found that Epstein-Barr virus proteins viral-caspid antigen-immunoglobulin A (VCA/IgA) and early antigen-immunoglobulin A (EA/IgA), but not Epstein-Barr virus-deoxyribonucleic acid, had a closed association with nasopharyngeal carcinoma metastasis. However, Epstein-Barr virus hallmarks failed to associate with other nasopharyngeal carcinoma characteristics. Furthermore, we confirmed that matrix metalloproteinase 9 existed in circulating tumor cells and expressed most in mesenchymal circulating tumor cells. In addition, matrix metalloproteinase 9-expressed extent in hybrid circulating tumor cells is somewhat different from epithelial and mesenchymal circulating tumor cells in matrix metalloproteinase 9-positive circulating tumor cells. Nevertheless, matrix metalloproteinase 9 had no relationship with other nasopharyngeal carcinoma characteristics. Finally, our results showed that circulating tumor cells were decreased in patients after therapies. carcinoma metastasis. Of note, decreased circulating tumor cells indicated a favorable curative effect in nasopharyngeal carcinoma patients.

In this study, we investigated the relationship between the epithelial–mesenchymal transition phenotype of circulating tumor cells (CTCs) and distant metastasis in breast cancer patients. We analyzed the expression of epithelial (epithelial cell adhesion molecule, cytokeratin [CK]8, CK18 and CK19) and mesenchymal (vimentin and TWIST1) markers in CTCs from a large cohort of Chinese breast cancer patients (N=1083) using Canpatrol™ CTC assays. We identified CTCs in 84.9% (920/1083) of the breast cancer patients enrolled in this study. Among these 920 patients, 547 showed epithelial CTCs, 793 showed biphenotypic CTCs and 516 showed mesenchymal CTCs. Receiver operating characteristic (ROC) curves demonstrated circulation of both biphenotypic and mesenchymal CTCs (area under ROC curve value: 0.728; sensitivity: 68.7% and specificity: 71.6%) in patients was associated with distant metastasis. These findings demonstrate that the epithelial–mesenchymal transition phenotype of CTCs is a potential biomarker predictive of distant metastasis in breast cancer.

Transporter-facilitated arsenite extrusion is the major pathway of arsenic resistance within bacteria. So far only two types of membrane-bound transporter proteins, ArsB and ArsY (ACR3), have been well studied, although the arsenic transporters in bacteria display considerable diversity. Utilizing accumulated genome sequence data, we searched arsenic resistance (ars) operons in about 2,500 bacterial strains and located over 700 membrane-bound transporters which are encoded in these operons. Sequence analysis revealed at least five distinct transporter families, with ArsY being the most dominant, followed by ArsB, ArsP (a recently reported permease family), Major Facilitator protein Superfamily (MFS) and Major Intrinsic Protein (MIP). In addition, other types of transporters encoded in the ars operons were found, but in much lower frequencies. The diversity and evolutionary relationships of these transporters with regard to arsenic resistance will be discussed.