GCC2 as a New Early Diagnostic Biomarker for Non-Small Cell Lung Cancer.

No specific markers have been identified to detect non-small cell lung cancer (NSCLC) cell-derived exosomes circulating in the blood. Here, we report a new biomarker that distinguishes between cancer and non-cancer cell-derived exosomes. Exosomes isolated from patient plasmas at various pathological stages of NSCLC, NSCLC cell lines, and human pulmonary alveolar epithelial cells isolated using size exclusion chromatography were characterized. The GRIP and coiled-coil domain-containing 2 (GCC2) protein, involved in endosome-to-Golgi transport, was identified by proteomics analysis of NSCLC cell line-derived exosomes. GCC2 protein levels in the exosomes derived from early-stage NSCLC patients were higher than those from healthy controls. Receiver operating characteristic curve analysis revealed the diagnostic sensitivity and specificity of exosomal GCC2 to be 90% and 75%, respectively. A high area under the curve, 0.844, confirmed that GCC2 levels could effectively distinguish between the exosomes. These results demonstrate GCC2 as a promising early diagnostic biomarker for NSCLC.

Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte.

Direct conversion is a powerful approach to safely generate mature neural lineages with potential for treatment of neurological disorders. Astrocytes play a crucial role in neuronal homeostasis and their dysfunctions contribute to several neurodegenerative diseases. Using a single-cell approach for precision, we describe here a robust method using optimized DNA amounts for the direct conversion of mouse fibroblasts to astrocytes. Controlled amount of the reprogramming factors Oct4, Sox2, Klf4 and cMyc was directly delivered into a single fibroblast cell. Consequently, 2500 DNA molecules, no more or less, were found to be the optimal amount that dramatically increased the expression levels of the astrocyte-specific markers GFAP and S100b and the demethylation gene TET1, the expression of which was sustained to maintain astrocyte functionality. The converted astrocytes showed glutamate uptake ability and electrophysiological activity. Furthermore, we demonstrated a potential mechanism whereby fibroblast was directly converted into astrocyte at a single-cell level; this was achieved by activating BMP2 pathway through direct binding of Sox2 protein to BMP2 gene. This study suggests that nanotechnology for directly injecting plasmid DNAs into cell nuclei may help understand such a conversion at single-cell level.

Spatio-temporally controlled transfection by quantitative injection into a single cell.

Transfection-based cellular control has been widely used in biology; however, conventional transfection methods cannot control spatio-temporal differences in gene expression or the quantity of delivered materials such as external DNA or RNA. Here, we present a non-viral and spatio-temporally controlled transfection technique of a quantitative injection into a single cell. DNA was quantitatively injected into a single cell at a desired location and time, and the optimal gene delivery and expression conditions were determined based on the amount of the delivered DNA and the transfection efficacy. Interestingly, an injection of 1500 DNAs produced an about average 30% gene expression efficiency, which was the optimal condition, and gene expression was sustained for more than 14 days. In a single cell, fluorescent intensity and polymerase chain reaction (PCR) results were compared for the quantity of gene expression. The high coincidence of both results suggests that the fluorescence intensity can reveal gene expression level which was investigated by PCR. In addition, 3 multiple DNA genes were successfully expressed in a single cell with different ratio. Overall, these results demonstrate that spatio-temporally controlled transfection by quantitative transfection is a useful technique for regulating gene expression in a single cell, which suggests that this technique may be used for stem cell research, including the creation of induced pluripotent stem (iPS) cells.