Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics‐based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.

While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue–tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro .

Ebola virus, for which we lack effective countermeasures, causes hemorrhagic fever in humans, with significant case fatality rates. Lack of experimental human models for Ebola hemorrhagic fever is a major obstacle that hinders the development of treatment strategies. Here, we model the Ebola hemorrhagic syndrome in a microvessel-on-a-chip system and demonstrate its applicability to drug studies. Luminal infusion of Ebola virus-like particles leads to albumin leakage from the engineered vessels. The process is mediated by the Rho/ROCK pathway and is associated with cytoskeleton remodeling. Infusion of Ebola glycoprotein (GP 1,2) generates a similar phenotype, indicating the key role of GP 1,2 in this process. Finally, we measured the potency of a recently developed experimental drug FX06 and a novel drug candidate, melatonin, in phenotypic rescue. Our study confirms the effects of FX06 and identifies melatonin as an effective, safe, inexpensive therapeutic option that is worth investigating in animal models and human trials.

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The cell has a three-dimensional (3D) structure and its spatial arrangement is often very important to molecular mechanisms essential for life. In order to visualize 3D morphologies of cells, confocal laser imaging was developed. 1 The method is, however, only applicable to fluorescence-probed molecules, 2 which limits the observable number of molecules, and such artificial probing sometime perturbs normal molecular mechanisms. Cotte et al. applied holographic and tomographic irradiation to microscopy and finally innovated a threedimensional computed holographic and tomographic (HT) laser microscope. 3 The laser beam that penetrates the cell at an angle experiences a delay in the phase of its beam, which is magnified and overlayed with reference beam to make a holographic image. The holograms at various angles then deconvoluted by tomographic algorithms to create a precise 3D cell image. The 3D-HT microscope can visualize 3D morphological aspects by contrasting refractive indexes observed by the laser monochromatic wavelength, making staining unnecessary.We have developed live single-cell mass spectrometry, 4-7 in which the contents of a single cell, usually picoliter level or less, are sucked by a nanospray tip (a sort of glass capillary needle) and fed directly into a mass spectrometer after the addition of an ionization solvent to the rear end of the tip. In this method, the exact amount sucked is unclear because it is such a tiny volume. Furthermore, 3D spatial location and identity of the contents are also ambiguous. Through the combination of these two techniques, 3D-HT microscopy and live single-cell mass spectrometry, greater 3D spatial resolution (X-Y-axis 0.18 μm and Z-axis 0.33 μm) and improved quantitative single-cell analysis is expected. The first trial of this combination and its results are documented in this paper, and we think nextgeneration live single-cell mass spectrometry is quite promising.Human hepatocellular carcinoma cell line (HepG2) was cultured in Dulbecco's modified Eagle medium in addition to 10% fetal calf serum (FBS), 100 mg/mL penicillin, and 100 mg/mL streptomycin G in 35 mm glass bottom dishes at 37 C and 5% CO2. HepG2 cells were positioned under the HT laser microscope, and the HT scan took 2 s to acquire one 3D image. Figure 1 shows the schematic principle of the HT laser microscope (3D Cell Explorer, Nanolive, SA, Switzerland). Fig. 1 Schematic of live single-cell mass spectrometry with quantitation by holographic and tomographic laser microscopy. The laser beam is split into a reference beam (going down to the CCD camera) and an observation beam that irradiates the cell at 45 degree angle. A micromanipulator was setup next to microscope to allow precise suction with a nanospray tip. The sucked cellular matter was then blasted through the mass spectrometer.

The dynamics of a cell is always changing. Cells move, divide, communicate, adapt, and are always reacting to their surroundings non-synchronously. Currently, single-cell metabolomics has become the leading field in understanding the phenotypical variations between them, but sample volumes, low analyte concentrations, and validating gentle sample techniques have proven great barriers toward achieving accurate and complete metabolomics profiling. Certainly, advanced technologies such as nanodevices and microfluidic arrays are making great progress, and analytical techniques, such as matrix-assisted laser desorption ionization (MALDI), are gaining popularity with high-throughput methodology. Nevertheless, live single-cell mass spectrometry (LCSMS) values the sample quality and precision, turning once theoretical speculation into present-day applications in a variety of fields, including those of medicine, pharmaceutical, and agricultural industries. While there is still room for much improvement, it is clear that the metabolomics field is progressing toward analysis and discoveries at the single-cell level.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC−MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC−MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC−MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC−MS to investigate pharmacokinetics at the single-cell level.