Single Cell Analysis with Probe ESI-Mass Spectrometry: Detection of Metabolites at Cellular and Subcellular Levels

Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 μm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell “omics”

SW inhibits cell scratch wound healing.

<p><i>In vitro</i> scratch assay was used to evaluate the effect of SW on the migration of HepG2 (A), MDA-MB-231 (B) and T24 cells (C). Representative images were displayed in left panel; quantification of the data in left panel was shown in right panel, shown were composite results of three independently experiments with triplicate parallel samples. The migration index represents migration speed in relative to control group. Columns, mean; bars, SD.</p

SW inhibits cell migration.

<p>HepG2 (A), T24 (B) and MDA-MB-231 (C) cells were seeded into the transwell insert in the presence of indicated doses of SW for 12 h. Cells reaching the bottom side of the transwell membrane were stained and representative images were displayed in the upper panel. Scale bar, 200 μm. The data in left panel were quantified and shown in the middle panel, and the cell viability was reflected by cell confluence rate in the right panel. Shown were composite results of two independently experiments. Columns, mean; bars, SD; NS, not statistically significant.</p

SW promotes cell adhesion.

<p>Left panels: HepG2 (A), MDA-MB-231 (B) and T24 cells (C) were pretreated with indicated doses of SW for 15 or 30 min before seeding into the matrigel-coated wells, and 2 h later, adhered cells were counted after removing the floating cells by PBS. Representative images were displayed. Scale bar, 200 μm. Right panels: quantification of the data in the left panel, shown were composite results of three independently experiments with triplicate. Columns, mean; bars, SD.</p

SW increases size and fluorescence intensity of focal adhesion.

<p>(A) HepG2 and T24 cells were treated with SW (3.5 μg/μl) for indicated times and then immuno-stained with F-actin (FITC-phalloidin) and vinculin (red). Nuclei were counterstained by DAPI (blue). Representative images were displayed. Scale bar, 100 μm. (B) The area of adhesion sites per cell (where F-actin and vinculin merged). Shown were composite results of two independently experiments (n = 50 cells). Columns, mean; bars, SD. (C) The fluorescence intensity of vinculin in the adhesion patches per cell. Shown were composite results of two independently experiments (n = 50 cells). Columns, mean; bars, SD.</p

Sarsaparilla (<i>Smilax Glabra</i> Rhizome) Extract Inhibits Migration and Invasion of Cancer Cells by Suppressing TGF-β1 Pathway

<div><p>Sarsaparilla, also known as <i>Smilax Glabra</i> Rhizome (SGR), was shown to modulate immunity, protect against liver injury, lower blood glucose and suppress cancer. However, its effects on cancer cell adhesion, migration and invasion were unclear. In the present study, we found that the supernatant of water-soluble extract from SGR (SW) could promote adhesion, inhibit migration and invasion of HepG2, MDA-MB-231 and T24 cells <i>in vitro</i>, as well as suppress metastasis of MDA-MB-231 cells <i>in vivo</i>. Results of F-actin and vinculin dual staining showed the enhanced focal adhesion in SW-treated cells. Microarray analysis indicated a repression of TGF-β1 signaling by SW treatment, which was verified by real-time RT-PCR of TGF-β1-related genes and immunoblotting of TGFBR1 protein. SW was also shown to antagonize TGF-β1-promoted cell migration. Collectively, our study revealed a new antitumor function of Sarsaparilla in counteracting invasiveness of a subset of cancer cells by inhibiting TGF-β1 signaling.</p></div

SW inhibits cell invasion <i>in vitro</i> and metastasis <i>in vivo</i>.

<p>(A) The transwell chambers were pre-coated with 60 μl matrigel dilution (1:4 in serum-free medium) before seeding cells. Cells were incubated with indicated doses of SW for 36 h before staining. Representative images were displayed in the upper panel. Scale bar, 200 μm. The data of migration and cell viability were quantified and shown respectively in lower panel, shown were composite results of two independently experiments with triplicate. Columns, mean; bars, SD; NS, not statistically significant. (B) Left panel: representative pictures of H&E stained lung tissues in PBS- and SW-treated group. Paraffin-embedded lungs were sectioned at 30-μm intervals and > 10 MDA-MB-231 cancer cells were identified as metastatic foci. Magnification, 4× (upper) and 20× (lower). Right panel: quantification of lung metastatic foci in PBS- and SW-treated mice group (n = 8 for each group). Columns, mean; bars, SD.</p

SW abrogates TGF-β1-induced boost in migration.

<p>HepG2 (A), MDA-MB-231 (B) and T24 (C) cells were seeded into the transwell inserts in the presence of TGF-β1 plus SW. 12 h later, Cells were stained and pictured. Representative images were displayed in the left panel. Scale bar, 100 μm. The data of migration were quantified and shown in the middle panel. The cell viability was reflected by cell confluence rate in the right panel. Shown was composite results of two independently experiments with triplicate. Columns, mean; bars, SD; NS, not statistically significant.</p