Single HeLa Cells Research Articles

Fluorescent visual quantitation of cell-secreted sialoglycoconjugates by chemoselective recognition and hybridization chain reaction.

Sialic acid (SA), usually located at the termini of glycan chains, is one of the most important monosaccharide blocks for glycosylation of proteins. The expression level of sialoglycoconjugates (SiaGCs) in cellular secretome is of great significance in diagnosis of tumor malignancy. This work developed a fluorescent visual method for the detection of SiaGCs secreted from living cells by a boronic acid modified chip based chemoselective recognition and hybridization chain reaction. The cell-secreted SiaGCs, which were labeled with the azide group through a metabolic labeling technique during cell culture, were captured by the chip through chemoselective recognition of boronic acid toward SA. After further conjugating the azide group with an alkyne modified DNA probe, the captured SiaGCs could be conveniently endowed with the amplified fluorescent signal through a hybridization chain reaction of a pair of dye-labeled DNA hairpins, which led to a quantitative imaging method for detection of SiaGCs. The average amount of metabolically labeled SiaGCs secreted from a single HeLa cell and MCF-7 cell was 2.18 × 10-17 and 3.98 × 10-17 mol, respectively. The proposed method could be utilized to monitor the variation of the secreted SiaGCs during drug treatment, providing a useful tool for investigating the glycosylation and glycan-related biological processes.

Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry.

Low-molecular-weight (LMW) thiols are important small molecules that regulate or maintain redox homeostasis in physiological and pathological processes. Assessing the concentrations of LMW thiols in biological systems may provide valuable information regarding physiological processes and the early diagnosis of some diseases. Here, we developed a method to simultaneously determine the concentrations of multiple LWM thiols in single cells by chemical derivatization assisted liquid chromatography-mass spectrometry (LC-MS). In this method, we synthesized a pair of stable isotope reagents, N-(acridin-9-yl)-2-bromoacetamide (AYBA) and N-(1,2,3,4-[2H4]-acridin-9-yl)-2-bromoacetamide ([2H4]AYBA). AYBA was used to derivatize LWM thiols in human cervical cancer (HeLa) cells, while [2H4]AYBA was used to derivatize standard LWM thiols to prepare internal standards for the LC-MS method development. The proposed AYBA derivatization greatly enhanced the detection sensitivity of LWM thiols by LC-MS, and thereby achieved the simultaneous detection of multiple LWM thiols by LC-MS in ∼1000 HeLa cells. Finally, the developed method was successfully utilized for the quantitative analysis of multiple LWM thiols in a single HeLa cell and the content changes of LWM thiols in a single HeLa cell before and after oxidative stress treatment. Accordingly, six LMW thiols were detected, including cysteamine, cysteine, glutathione, homocysteine, hydrogen sulfide, and pantetheine.

Understanding flow dynamics, viability and metastatic potency of cervical cancer (HeLa) cells through constricted microchannel

To understand the burgeoning challenges of metastasis, a microchannel of 35 μm diameter, constricted to 7 μm for a distance of 200 μm in a total length of 3 mm, was designed and fabricated using a mask aligner made of polydimethylsiloxane (PDMS) to mimic in vivo capillaries. A thin glass cover-slide was mounted on top to monitor the motion of single or aggregated malignant HeLa cells (size 17–30 μm) microscopically through the constricted microchannel at a constant flow rate of 30 μl/h. Quantitative deconvolution of high-speed videographs of a single cell of 30 μm revealed cellular deformation while passing through constriction, having elongation index, average transit velocity and entry time of 2.67, 18 mm/s and 5.1 ms, respectively. Morphological analysis of live and apoptotic cells by dual staining with Acridine Orange/Ethidium Bromide demonstrated retention of a significant viable cell population after exit through the constriction and a viability index of 50% was quantified by dye exclusion assay. The cumulative data for microfluidic parameters, morphology and relevant metastatic MMP2 gene expression efficiency measured by real-time polymerase chain reaction revealed retention of virulence potency that could possibly cause metastasis, would be beneficial in developing futuristic MEMS device for cancer theranostics.

Integrated Proteome Analysis Device for Fast Single-Cell Protein Profiling

In our previous work, we have demonstrated an integrated proteome analysis device (iPAD-100) to analyze proteomes from 100 cells. (1) In this work, for the first time, a novel integrated device for single-cell analysis (iPAD-1) was developed to profile proteins in a single cell within 1 h. In the iPAD-1, a selected single cell was directly sucked into a 22 μm i.d. capillary. Then the cell lysis and protein digestion were simultaneously accomplished in the capillary in a 2 nL volume, which could prevent protein loss and excessive dilution. Digestion was accelerated by using elevated temperature with ultrasonication. The whole time of cell treatment was 30 min. After that, single-cell digest peptides were transferred into an LC column directly through a true zero dead volume union, to minimize protein transfer loss. A homemade 22 μm i.d. nano-LC packing column with 3 μm i.d. ESI tip was used in the device to achieve ultrasensitive detection. A 30 min elution program was applied to analysis of the single-cell proteome. Therefore, the total time needed for a single-cell analysis was only 1 h. In an analysis of 10 single HeLa cells, a maximum of 328 proteins were identified in one cell by using an Orbitrap Fusion Tribrid MS instrument, and the detection limit was estimated at around 1.7-170 zmol. Such a sensitivity of the iPAD-1 was ∼120-fold higher than that of our previously developed iPAD-100 system. (1) Prominent cellular heterogeneity in protein expressive profiling was observed. Furthermore, we roughly estimated the phases of the cell cycle of tested HeLa cells by the amount of core histone proteins.

T-Probe: An Integrated Microscale Device for Online In Situ Single Cell Analysis and Metabolic Profiling Using Mass Spectrometry.

The exploration of single cells reveals cell heterogeneity and biological principle of cellular metabolism. Although a number of mass spectrometry (MS) based single cell MS (SCMS) techniques have been dedicatedly developed with high efficiency and sensitivity, limitations still exist. In this work, we introduced a microscale multifunctional device, the T-probe, which integrates cellular contents extraction and immediate ionization, to implement online in situ SCMS analysis at ambient conditions with minimal sample preparation. With high sensitivity and reproducibility, the T-probe was employed for MS analysis of single HeLa cells under control and anticancer drug treatment conditions. Intracellular species and xenobiotic metabolites were detected, and changes of cellular metabolic profiles induced by drug treatment were measured. Combining SCMS experiments with statistical data analyses, including Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) and two-sample t-test, we provided biological insights into cellular metabolic response to drug treatment. Online MS/MS analysis was conducted at single cell level to identify species of interest, including endogenous metabolites and the drug compound. Using the T-probe SCMS technique combined with comprehensive data analyses, we provide an approach to understanding cellular metabolism and evaluate chemotherapies at the single cell level.

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS.

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC‐MS

AbstractWe report on the quantitative proteomic analysis of single mammalian cells. Fluorescence‐activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC‐MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single‐cell proteomics platform can be used to differentiate cell types from enzyme‐dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

A compact and low-cost laser induced fluorescence detector with silicon based photodetector assembly for capillary flow systems

A compact and low-cost laser induced fluorescence (LIF) detector based on confocal structure for capillary flow systems was developed and applied for analysis of Her2 protein on single Hela cells. A low-power and low-cost 450 nm laser diode (LD) instead of a high quality laser was used as excitation light source. A compact optical design together with shortened optical path length improved the optical efficiency and detection sensitivity. A superior silicon based photodetector assembly was used for fluorescence detection instead of a photomultiplier (PMT). The limit of detection (LOD) for fluorescein sodium was 3 × 10–12 M or 165 fluorescein molecules in detection volume measured on a homemade capillary electroosmotic driven (EOD)-LIF system, which was similar to commercial LIFs. Compared to commercial LIFs, the whole volume of our LIF was reduced to 1/2–1/3, and the cost was less than 1/3 of them.

Dynamics and mechanisms of intracellular calcium waves elicited by tandem bubble-induced jetting flow

One of the earliest events in cellular mechanotransduction is often an increase in intracellular calcium concentration associated with intracellular calcium waves (ICWs) in various physiologic or pathophysiologic processes. Although cavitation-induced calcium responses are believed to be important for modulating downstream bioeffects such as cell injury and mechanotransduction in ultrasound therapy, the fundamental mechanisms of these responses have not been elucidated. In this study, we investigated mechanistically the ICWs elicited in single HeLa cells by the tandem bubble-induced jetting flow in a microfluidic system. We identified two distinct (fast and slow) types of ICWs at varying degrees of flow shear stress-induced membrane deformation, as determined by different bubble standoff distances. We showed that ICWs were initiated by an extracellular calcium influx across the cell membrane nearest to the jetting flow, either primarily through poration sites for fast ICWs or opening of mechanosensitive ion channels for slow ICWs, which then propagated in the cytosol via a reaction-diffusion process from the endoplasmic reticulum. The speed of ICW (CICW ) was found to correlate strongly with the severity of cell injury, with CICW in the range of 33 μm/s to 93 μm/s for fast ICWs and 1.4 μm/s to 12 μm/s for slow ICWs. Finally, we demonstrated that micrometer-sized beads attached to the cell membrane integrin could trigger ICWs under mild cavitation conditions without collateral injury. The relation between the characteristics of ICW and cell injury, and potential strategies to mitigate cavitation-induced injury while evoking an intracellular calcium response, may be particularly useful for exploiting ultrasound-stimulated mechanotransduction applications in the future.

Size-Controllable Gold Nanopores with High SERS Activity.

Nanopore structures have been successfully employed in next-generation DNA sequencing. For more complicated protein which normally contains 20 different amino acids, identifying the fluctuation of ionic current caused by different amino acids appears inadequate for protein sequencing. Therefore, it is highly desirable to develop size-controllable nanopores with optical activity that can provide additional structural information. Herein, we discovered the novel nanopore properties of the self-assembled ultramicroelectrodes originally developed by Bard and co-workers. Using a slightly modified method, the self-assembly of 7 ± 1 nm gold nanoparticles (AuNPs) can be precisely controlled to form a gold nanoporous sphere (GPS) on the tip of a glass capillary. Different dithiol linker molecules (1,3-propanedithiol, C3; 1,6-hexanedithiol, C6; and 1,9-nonanedithiol, C9) reproducibly led to rather similar nanopore sizes (5.07 ± 0.02, 5.13 ± 0.02, and 5.25 ± 0.01 nm), respectively. The GPS nanostructures were found to exhibit high ionic current rectification as well as surface-enhanced Raman scattering (SERS) activity due to the presence of nanopores and numerous "hot spots" among the cross-linked AuNPs on the surface of GPS. The rectification effect of the small nanopores was observed even under high concentration of electrolyte (290 mM), along with SERS enhancement factors well above 1 × 105. The GPS nanostructures were successfully applied for SERS-based detection of glutathione from a single HeLa cell.

Point-of-Care Assay of Telomerase Activity at Single-Cell Level via Gas Pressure Readout.

Detection of telomerase activity at the single-cell level is one of the central challenges in cancer diagnostics and therapy. Herein, we describe a facile and reliable point-of-care testing (POCT) strategy for detection of telomerase activity via a portable pressure meter. Telomerase primer (TS) was immobilized onto the surface of magnetic beads (MBs), and then was elongated to a long single-stranded DNA by telomerase. The elongated (TTAGGG)n repeat unit hybridized with several short PtNP-functionalized complementary DNA (PtNPs-cDNA), which specifically enriched PtNPs onto the surfaces of magnetic beads (MBs), which were separated using a magnet. Then, nanoparticle-catalyzed gas-generation reaction converted telomerase activity into significant change in gas pressure. Because of the self-amplification of telomerase and enrichment by magnetic separation, the diluted telomerase equivalent to a single HeLa cell was facilely detected. More importantly, the telomerase in the lysate of 1 HeLa cell can be reliably detected by monitoring change in gas pressure, indicating that it is feasible and possible to study differences between individual cells. The difference in relative activity between different kinds of cancer cells was easily and sensitively studied. Study of inhibition of telomerase activity demonstrated that our method has great potential in screening of telomerase-targeted antitumor drugs as well as in clinical diagnosis.

A label-free sensitive method for membrane protein detection based on aptamer and AgNCs transfer

Recently, membrane proteins have been considered as candidate cancer biomarkers and drug targets, due to their important roles in numerous physiological processes. Therefore, a facile, sensitive and quantitative detection of the membrane proteins is crucial for better understanding their roles in cancer cells and further validating their function in clinical research. We report a highly facile and sensitive detection method for membrane proteins on living cells in situ based on membrane protein-triggered release of cytosine (C)-rich single-stranded DNA (ssDNA) sequences, and the subsequent silver nanoclusters (AgNCs) transfer from polymer to C-rich ssDNA. The high-quantum yield and stable DNA-AgNCs allow the accurate detection of membrane proteins with facile operations and a common fluorescence spectrophotometer. The detection of protein tyrosine kinase-7 (PTK7), a membrane protein model, displays a response range from 30pM to 2nM with a detection limit of 12pM. The expression of PTK7 on single Hela cell and CCRF-CEM cell was calculated to be 7.5 × 10−19mol and 1.8 × 10−18mol, respectively. Given the simple and facile operation of this method, this detection platform can be applied as a universal strategy for ultrasensitive detection of membrane protein on cell in situ.

Abstract 514: Genetic load in cancer cell populations

Abstract Populations accumulate deleterious mutations that have yet to be removed by natural selection. This mutated portion of the population that does not contribute to its fitness is called the genetic load and is proportional to the deleterious mutation rate. Given the genomic instability of cancer cells, the genetic load could be substantial if the instability indeed leads to loss of cell fitness. We hypothesize that because aneuploidy and copy number variations (CNVs) are the most common forms of genome instability, their fitness consequences may determine the genetic load in cancer cells. To test this hypothesis, we randomly selected single cells from a HeLa cell line and measured the cell growth rate via daily cell counting and an MTT cell proliferation assay. We then measured the genetic changes and resulting growth rate variation. We observed rapid generation of heterogeneity in the growth rate within the population. The growth rate of different progeny cells was stable indicating that it is genetically determined. Next, using whole genome sequencing of recently descended clones of a single HeLa cell, we found that cells with fewer newly acquired CNVs have higher proliferation rates vice versa. This indicated that CNV accumulation decreased cell fitness systematically. We next estimated the rate of CNV mutation by measuring the growth rate of two daughter cells from single proliferation and found that there is approximately 1 deleterious mutation in every 4 cell divisions. We speculated that due to a high, deleterious CNV mutation rate, tumor cells inevitably accumulate deleterious CNVs and a large percentage of tumor cells are genetically defective. Accordingly, we observed that the average growth rate of tumor cell populations decreased in the short term since defective cells accumulated in the population and the variation of cellular growth rates within the population increased. By modeling the process of mutation accumulation and measuring cell growth rate, we estimated that the deleterious mutation rate in HeLa cells is about 0.26-0.31 per cell division, and that HeLa cells reduce roughly 5%-6% of fitness for every cell division. The observations of a high proliferation rate and high genetic load in this representative tumor cell line indicates a “high risk, high reward” evolution strategy for tumor cells and suggests that increasing the level of genomic instability may cause the meltdown of tumor cell population by forcing cells to accelerate the cell cycle. Citation Format: Yuezheng Zhang, Xu Shen, Yawei Li, Tianqi Zhu, Yong Tao, Tao Li, Di Wang, Xueying Li, Qin Ma, Xuemei Lu, Hurng-Yi Wang, Chung-I Wu. Genetic load in cancer cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 514. doi:10.1158/1538-7445.AM2017-514

A localized chemical pulse generator for the development of a microfluidic cell-based biosensor

Cell-based biosensor (CBB) has shown potential to provide a rapid and sensitive measurement means for monitoring specific biological substances. However, the detecting frequency and reusability remain a challenge for further application of CBB. In this paper, a microfluidic cell-based biosensor (μCBB) was developed, which consisted of a localized millisecond chemical pulse as sample introduction for pulsing individual cells without interfering with adjacent cells and single HeLa cells expressing endogenous P2Y receptors as the sensing elements. Gated injection of pinched flow was used to generate millisecond ATP pulses with predefined width. By integrating localized ATP pulse with predefined width, multiple sensing elements could be used to detect ATP pulses without impact on their functionality. Thus, the detecting frequency of the developed μCBB is multiplied by the number of available cells in comparison to that reported previously. We expect the developed method to be potentially useful to enhance the usability of CBB for biological and biomedical analysis.

Quantitative electrophysiological monitoring of anti–histamine drug effects on live cells via reusable sensor platforms

We demonstrated the quantitative electrophysiological monitoring of histamine and anti–histamine drug effects on live cells via reusable sensor platforms based on carbon nanotube transistors. This method enabled us to monitor the real–time electrophysiological responses of a single HeLa cell to histamine with different concentrations. The measured electrophysiological responses were attributed to the activity of histamine type 1 receptors on a HeLa cell membrane by histamine. Furthermore, the effects of anti–histamine drugs such as cetirizine or chlorphenamine on the electrophysiological activities of HeLa cells were also evaluated quantitatively. Significantly, we utilized only a single device to monitor the responses of multiple HeLa cells to each drug, which allowed us to quantitatively analyze the antihistamine drug effects on live cells without errors from the device-to-device variation in device characteristics. Such quantitative evaluation capability of our method would promise versatile applications such as drug screening and nanoscale bio sensor researches.

Nanoscaled Porphyrinic Metal-Organic Frameworks for Electrochemical Detection of Telomerase Activity via Telomerase Triggered Conformation Switch.

In this work, we designed a nanoscaled porphyrinic metal-organic framework (PorMOF) with iron porphyrin as linker and zirconium ion as node for electrochemical detection of telomerase activity. The as-prepared PorMOF was characterized with scanning electron microscopy, powder X-ray diffraction, and spectroscopic techniques and demonstrated excellent electrocatalytic activity toward O2 reduction. Sequentially, the functionalization of PorMOF with streptavidin results in a water-stable electrochemical tracer for detection of telomerase. Upon the telomerase-triggered extension, the assistant DNA 1 (aDNA1)-assistant DNA 2 (aDNA2) duplex could switch into a hairpin structure, and thus, the aDNA2 was released and then hybridized with the capture DNA. Therefore, the PorMOF@SA tracer could be introduced on the electrode surface via biotin-streptavidin recognition, leading to the strong electrochemical signal for readout. The developed approach displayed desirable dynamic range and limitation of detection down to 30 HeLa cells mL-1. The telomerase activity was 2.2 × 10-11 IU in a single HeLa cell with good reproducibility and stability. The nanoscaled porphyrinic MOF provided a powerful platform for electrochemical signal transduction and had a promising application in the determination of various biomolecules.

Scissor‐Like Chiral Metamolecules for Probing Intracellular Telomerase Activity

A DNA‐driven gold (Au) heterodimer for intracellular telomerase detection is fabricated. The highly biocompatible and intracellularly stable probe shows an active chiroptical property in the visible region, due to the scissor‐like configuration formed by prolate nanoparticles. Importantly, the telomerase activity is specifically quantified using circular dichroism intensity in situ after internalization of the heterodimer into cancer cells. Moreover, the results clearly illustrate that this method has a remarkable linear range from 0.8 × 10−12 to 32 × 10−12 IU, and the limit of detection for telomerase activity is 1.7 × 10−15 IU in a single HeLa cell. This strategy paves the way for chirality‐based ultrasensitive detection of intracellular cancer markers.

Focal adhesions control cleavage furrow shape and spindle tilt during mitosis.

The geometry of the cleavage furrow during mitosis is often asymmetric in vivo and plays a critical role in stem cell differentiation and the relative positioning of daughter cells during development. Early observations of adhesive cell lines revealed asymmetry in the shape of the cleavage furrow, where the bottom (i.e., substrate attached side) of the cleavage furrow ingressed less than the top (i.e., unattached side). This data suggested substrate attachment could be regulating furrow ingression. Here we report a population of mitotic focal adhesions (FAs) controls the symmetry of the cleavage furrow. In single HeLa cells, stronger adhesion to the substrate directed less ingression from the bottom of the cell through a pathway including paxillin, focal adhesion kinase (FAK) and vinculin. Cell-cell contacts also direct ingression of the cleavage furrow in coordination with FAs in epithelial cells—MDCK—within monolayers and polarized cysts. In addition, mitotic FAs established 3D orientation of the mitotic spindle and the relative positioning of mother and daughter centrosomes. Therefore, our data reveals mitotic FAs as a key link between mitotic cell shape and spindle orientation, and may have important implications in our understanding stem cell homeostasis and tumorigenesis.

Investigating intercellular calcium waves by microfluidic gated pinched-flow

In this paper, a new microfluidic approach, termed as gated pinched-flow (GPF), was developed for investigating cell-to-cell signaling dynamics coupling with optical imaging. It takes both advantages of conventional hydrodynamic gating and focusing, enabling precise on-chip chemical stimulation or perfusion upon targeted single cells among cells populations with high temporal (<50ms) and spatial resolution. Theoretical model for GPF was first established and further validated by both numerical simulations and experiments. Stimulation of single HeLa cells was investigated to demonstrate the capability of the GPF for localized chemical stimulations of target single cells without interfering with adjacent cells. Guided by GPF, ATP-activated propagation of intercellular calcium waves (ICWs) among the seeded NIH-3T3 cells in the microchannel was then imaged with high repeatability. Inhibition investigations verified that those cell-to-cell calcium signals depended upon direct cytosolic transfer of molecules via gap junctions. The developed microfluidic method opens up a new avenue for cell-to-cell signaling studies and drug screening.

24 h observation of a single HeLa cell by impedance measurement and numerical modeling

Comparing to conventional examinations, a continuous impedance analysis of single cells provide more detailed electrical characterization information about their pathological condition in a period. In this work, we present the 24-h observations of the electrical characteristics of single HeLa (human cervix adenocarcinoma) cells using impedance measurement and modeling method. A microfluidic device includes three-micropillars structure and measurement electrodes are used to single HeLa cell capture and impedance measurement, respectively. An electrical circuit comprising cytoplasm resistance, cell membrane capacitance, medium resistance, medium capacitance and electrodes resistance is used to obtain the variation on the location, shape and configuration of single HeLa cell. According to experiment and modeling results, cell approached the bottom of the microchannel at 3–5h. At 5–15h, the impedance characteristics of the single HeLa cell changed with the cell shape. After 15h, cell membrane capacitance and cytoplasm resistance decreased due to cell membrane electroporation.