BackgroundA limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.MethodsThe 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.ResultsCTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).ConclusionEnriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined.

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The Wnt/β-catenin signaling is abnormally activated in the progression of hepatocellular carcinoma (HCC). BCL9 is an essential co-activator in the Wnt/β-catenin signaling. Importantly, BCL9 is absent from tumors originating from normal cellular counterparts and overexpressed in many cancers including HCC. But the mechanism for BCL9 overexpression remains unknown. Ample evidence indicates that hypoxia inducible factors (HIFs) play a role in the development of HCC. It was found in our study that BCL9 was overexpressed in both primary HCC and bone metastasis specimens; loss of BCL9 inhibited the proliferation, migration and angiogenesis of HCC; and that that hypoxia mechanically induced the expression of BCL9. BCL9 induction under the hypoxic condition was predominantly mediated by HIF-1α but not HIF2α. In vitro evidence from xenograft models indicated that BCL9 promoter/gene knockout inhibited HCC tumor growth and angiogenesis. Notably, we found that BCL9 and HIF-1α were coordinately regulated in human HCC specimen. The above findings suggest that hypoxia may promote the expression of BCL9 and associate with the development of HCC. Specific regulation of BCL9 expression by HIF-1α may prove to be an underlying crosstalk between Wnt/β-catenin signaling and hypoxia signaling pathways.

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The present study aimed to evaluate the functional connectivity (FC) in relevant cortex areas during simulated driving with distraction based on functional near-infrared spectroscopy (fNIRS) method. Twelve subjects were recruited to perform three types of driving tasks, namely, straight driving, straight driving with secondary auditory task, and straight driving with secondary visual vigilance task, on a driving simulator. The wavelet amplitude (WA) and wavelet phase coherence (WPCO) of the fNIRS signals were calculated in six frequency intervals: I, 0.6–2 Hz; II, 0.145–0.6 Hz; III, 0.052–0.145 Hz; IV, 0.021–0.052 Hz; and V, 0.0095–0.021 Hz, VI, 0.005–0.0095Hz. Results showed that secondary tasks during driving led to worse driving performance, brain activity changes, and dynamic configuration of the connectivity. The significantly lower WA value in the right motor cortex in interval IV, and higher WPCO values in intervals II, V, and VI were found with additional auditory task. Significant standard deviation of speed and lower WA values in the left prefrontal cortex and right prefrontal cortex in interval VI, and lower WPCO values in intervals I, IV, V, and VI were found under the additional visual vigilance task. The results suggest that the changed FC levels in intervals IV, V, and VI were more likely to reflect the driver’s distraction condition. The present study provides new insights into the relationship between distracted driving behavior and brain activity. The method may be used for the evaluation of drivers’ attention level.