Circulating tumour cells (CTCs) in blood circulation play an important role in cancer metastasis. CTCs are generally defined as the cells in circulating blood expressing the surface antigen EpCAM (epithelial cell adhesion molecule). Nevertheless, CTCs with a highly metastatic nature might undergo an epithelial-to-mesenchymal transition (EMT), after which their EpCAM expression is downregulated. In current CTC-related studies, however, these clinically important CTCs with high relevance to cancer metastasis could be missed due to the use of the conventional CTC isolation methodologies. To precisely explore the clinical significance of these cells (i.e., CD45neg/EpCAMneg cells), the high-purity isolation of these cells from blood samples is required. To achieve this isolation, the integration of fluorescence microscopic imaging and optically induced dielectrophoresis (ODEP)-based cell manipulation in a microfluidic system was proposed. In this study, an ODEP microfluidic system was developed. The optimal ODEP operating conditions and the performance of live CD45neg/EpCAMneg cell isolation were evaluated. The results demonstrated that the proposed system was capable of isolating live CD45neg/EpCAMneg cells with a purity as high as 100%, which is greater than the purity attainable using the existing techniques for similar tasks. As a demonstration case, the cancer-related gene expression of CD45neg/EpCAMneg cells isolated from the blood samples of healthy donors and cancer patients was successfully compared. The initial results indicate that the CD45neg/EpCAMneg nucleated cell population in the blood samples of cancer patients might contain cancer-related cells, particularly EMT-transformed CTCs, as suggested by the high detection rate of vimentin gene expression. Overall, this study presents an ODEP microfluidic system capable of simply and effectively isolating a specific, rare cell species from a cell mixture.

Growth arrest-specific 7 (Gas7) is preferentially expressed in the nervous system and plays an important role during neuritogenesis in mammals. However, the structure and function of Gas7 homologs have not been studied in nonmammalian vertebrates used as models. In this report, we identify a Gas7 gene in zebrafish that we termed zfGas7. The transcript of this gene was produced by canonical splicing, and its protein product contained a Fes/CIP4 homology and a coiled-coil domain. In early zebrafish embryos, RT-PCR analyses revealed that zfGas7 was initially expressed at 5.3 hr postfertilization (hpf), followed by an increase of expression at 10 hpf and further accumulation during somitogenesis at 48 hpf. Spatiotemporal analyses further showed that Gas7 mRNA was detected in the brain, somite, and posterior presomitic mesoderm regions during somitogenesis. At 36 hpf, zfGas7 mRNA was detected in the brain and somite but was later found only in neuronal clusters of the brain at 52 hpf. Gas7 knockdown with morpholino antisense oligonucleotides (Gas7MO) reduced the number of HuC-positive neurons in the trigeminal and statoacoustic ganglions and produced deformed phenotypes, such as flattening of the top of the head. Notably, the neuron reduction and deformed phenotypes observed in Gas7MO embryos were partially rescued by ectopic expression of Gas7. Because altered somitogenesis and pigmentation were also found in the morphants, the neuronal phenotypes observed likely are due to a general developmental delay of embryogenesis. These results indicate that Gas7 is expressed in neuronal cells but is not specifically required for neuronal development in vertebrates.

Conventional positive and negative selection-based circulating tumor cell (CTC) isolation methods might generally ignore metastasis-relevant CTCs that underwent epithelial-to- mesenchymal transition and suffer from a low CTC purity problem, respectively. To address these issues, we previously proposed a 2-step CTC isolation method integrating a negative selection CTC isolation and subsequent spheroid cell culture. In addition to its ability to isolate CTCs, more importantly, the spheroid cell culture used could serve as a cell culture model mimicking the process of new tumor tissue formation during cancer metastasis. Therefore, it is promising not only to selectively isolate metastasis-relevant CTCs but also to test the potential of cancer metastasis and thus the prognosis of disease. To explore these issues, experiments were performed. The key findings of this study demonstrated that the method was able to harvest both epithelial (E)- and mesenchymal (M)-type CTCs without selection bias. Moreover, both the M-type CTC count and the information obtained from the multidrug resistance-associated protein 2 (MRP2) and MRP5 gene expression analysis of the CTCs isolated via the 2-step CTC isolation method might be able to serve as prognostic factors for progression-free survival in head and neck squamous cell carcinoma.

The differentiation of bone marrow mesenchymal stem cells (MSCs) into osteoblasts is a crucial step during bone formation. However, the mechanisms regulating the early stages of osteogenic differentiation are not fully understood. In the present study, we found that growth-arrest specific gene 7b (Gas7b) was up-regulated during dexamethasone-induced differentiation of human MSCs (hMSCs) into osteoblasts. Knockdown of Gas7 using short-hairpin RNA decreased the expression of the osteogenic transcription factor Runx2 and its target genes alkaline phosphatase, type I collagen, osteocalcin (OC), and osteopontin. In addition, knockdown of Gas7 decreased matrix mineralization of dexamethasone-treated hMSCs in vitro. In contrast, ectopic expression of Gas7 isoforms a and b promoted gene expression associated with osteoblast differentiation and matrix mineralization, and also induced the mineralization of hMSCs in vitro. Furthermore, a gene reporter assay designed to monitor OC expression in hMSCs revealed that Runx2-dependent transcriptional activity was enhanced by over-expression of human Gas7 isoforms a and b. These findings reveal that Gas7 regulates the differentiation of hMSCs into osteoblasts by enhancing Runx2-dependent gene expression. ß

Efficient control of cell survival and cell proliferation is critical for the development of neuron cells. Earlier, we observed that growth arrest-specific gene 7 (Gas7) plays a role in controlling neuritogenesis in mammals. In the present study, we report that the Gas7b isoform is involved in controlling growth arrest and apoptosis of neuroblastoma cells in response to various stimuli. Accordingly, knockdown of Gas7b using small-hairpin RNA (shRNA) was shown to reduce apoptosis induced either by serum starvation or by the antineoplastic agents cisplatin and nocodazole in human neuroblastoma SH-SY5Y cells. Gas7b knockdown also enhanced the ability of the treated cells to form clones in response to cisplatin. On the other hand, forced expression of Gas7a or Gas7b isoform in mouse neuroblastoma Neuro2A cells, which express a defective Gas7 gene, rendered the cells proapoptotic and vulnerable to cisplatin-induced apoptosis. In addition, Neuro2A cells that overexpressed Gas7 showed a reduced ability to form clones. Overexpression of Gas7 produced similar but less extensive effects in nonneuronal HEK293 cells. Taken together, our observations suggest that Gas7b is involved not only in neuritogenesis but also in the regulation of neuronal cell death.