neu/erbB-2 amplification is an independent prognostic factor for risk of recurrence in ANN breast cancer. Women with tumors without neu/erbB-2 amplification have a good prognosis; aggressive therapy in this group is therefore difficult to justify. On the other hand, even with adjuvant chemotherapeutic treatment, women whose tumors exhibit neu/erbB-2 amplification have an increased risk of recurrence. We encourage a randomized trial to compare more aggressive adjuvant chemotherapy versus standard chemotherapy for ANN women whose tumors exhibit neu/erbB-2 amplification.
Serum and tissue DKK1 levels increased in a stepwise manner in multistep hepatocarcinogenesis and had prognostic significance. DKK1 plays a functional role in cell migration, invasion and tumour growth.
Since the discovery of neural stem cells (NSC) in the embryonic and adult mammalian central nervous system (CNS), there have been a growing numbers of tissue culture media and protocols to study and functionally characterize NSCs and its progeny in vitro. One of these culture systems introduced in 1992 is referred to as the Neurosphere Assay, and it has been widely used to isolate, expand, differentiate and even quantify NSC populations. Several years later because its application as a quantitative in vitro assay for measuring NSC frequency was limited, a new single-step semisolid based assay, the Neural Colony Forming Cell (NCFC) assay was developed to accurately measure NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e., their proliferative potential). The evolution and continued improvements made to these tissue culture tools will facilitate further advances in the promising application of NSCs for therapeutic use.
The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identify multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (i.e., suboptimal fluorescence signals, phototoxicity, and unphysiological neuronal activity). To overcome these issues, we develop a neuromedium called BrainPhys™ Imaging (BPI) in which we optimize the concentrations of fluorescent and phototoxic compounds. BPI is based on the formulation of the original BrainPhys medium. We benchmark available neuronal media and show that BPI enhances fluorescence signals, reduces phototoxicity and optimally supports the electrical and synaptic activity of neurons in culture. We also show the superior capacity of BPI for optogenetics and calcium imaging of human neurons. Altogether, our study shows that BPI improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting optimal neuronal viability and function.
The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology have improved exponentially in the last ten years. At the same time, advances in cellular reprogramming and organoid engineering have quickly expanded the use of human neuronal models in vitro. Altogether this creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identified multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (e.g., phototoxicity, suboptimal fluorescence signals, and unphysiological neuronal activity). To overcome these issues, we developed a new neuromedium, “BrainPhys™ Imaging”, in which we adjusted fluorescent and phototoxic compounds. The new medium is based on the formulation of the original BrainPhys medium, which we designed to better support the neuronal activity of human neurons in vitro1. We tested the new imaging-optimized formulation on human neurons cultured in monolayers or organoids, and rat primary neurons. BrainPhys Imaging enhanced fluorescence signals and reduced phototoxicity throughout the entire light spectrum. Importantly, consistent with standard BrainPhys, we showed that the new imaging medium optimally supports the electrical and synaptic activity of midbrain and human cortical neurons in culture. We also benchmarked the capacity of the new medium for functional calcium imaging and optogenetic control of human neurons. Altogether, our study shows that the new BrainPhys Imaging improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting cell viability and neuronal functions.
Recent reports have highlighted several parameters of the neurosphere culture or assay system which render it unreliable as a quantitative in vitro assay for measuring neural stem cell (NSC) frequency. The single-step semi-solid based assay, the Neural Colony Forming Cell (NCFC) assay is an assay which was developed to overcome some of the limitations of the neurospheres assay in terms of accurately measuring NSC numbers. The NCFC assay allows the discrimination between NSCs and progenitors by the size of colonies they produce (i.e. their proliferative potential). The NCFC assay and other improved tissue culture tools offer further advances in the promising application of NSCs for therapeutic use.
Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies in the world and is particularly prevalent in China and Southeast Asia. Although the risk factors are well known, the molecular mechanisms underlying hepatocarcinogenesis is unclear. Alteration of Wnt signaling pathway is one of the key events in HCC development and so we performed a low-density array (LDA) analysis on 38 pairs of HCC samples and their corresponding non-tumorous livers to examine the expression profiles of different Wnt signaling components. We found that, among the genes studied, Dishevelled 3 (Dvl3) was significantly overexpressed in HCCs as compared with the corresponding non-tumorous liver tissues. In addition, Dvl3 overexpression in HCC was significantly associated with presence of venous invasion (P = .021). The overexpression result was confirmed by expansion of the sample size using quantitative real-time PCR (q-PCR). Next, we investigated the effects of Dvl3 on the migratory and invasive abilities of HCC cells. We established stable shRNA Dvl3-knockdown clones in PLC/PRF/5 HCC cells and found that knockdown of Dvl3 suppressed the migratory and invasive abilities of HCC cells using transwell migration and invasion assays, respectively, suggesting an enhancing role of Dvl3 in HCC cell movement and cell invasion. Although cell proliferation assay in vitro showed no significant difference between the stable Dvl3 knockdown and non-target control PLC/PRF/5 cells, in vivo study in SCID mice showed that the stable Dvl3-knockdown cells resulted in a lower incidence of tumors formed and a smaller tumor size. Furthermore, since Dvl3 is an upstream regulator of the Wnt/β-catenin pathway, using the TOPFOP luciferase reporter assay, we found that transient transfection of Dvl3 in PLC/PRF/5 and BEL-7402 HCC cell lines enhanced the β-catenin-dependent transcriptional activity. In conclusion, upregulation of Dvl3 was frequent in human HCCs. Dvl3 enhanced cell migration and invasion in vitro and tumorigenicity in vivo in SCID mice, and activated canonical β-catenin pathway. It may play an oncogenic role in HCC development. (This study was funded by Hong Kong Research Grants Council CRF grants (RGC CRF HKU1/06C and HKU 7/CRG/09).) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2011-1074
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