Single-cell Microarray Research Articles

Novel mesothelin-targeted chimeric antigen receptor-modified UNKT cells are highly effective in inhibiting tumor progression

The design of chimeric antigen receptors (CAR) significantly enhances the antitumor efficacy of T cells. Although some CAR-T products have been approved by FDA in treating hematological tumors, adoptive immune therapy still faces many difficulties and challenges in the treatment of solid tumors. In this study, we reported a new strategy to treat solid tumors using a natural killer-like T (NKT) cell line which showed strong cytotoxicity to lyse 15 cancer cell lines, safe to normal cells and had low or no Graft-versus-host activity. We thus named it as universal NKT (UNKT). In both direct and indirect 3D tumor-like organ model, UNKT showed efficient tumor-killing properties, indicating that it could penetrate the microenvironment of solid tumors. In mesothelin (MSLN)-positive tumor cells (SKOV-3 and MCF-7), MSLN targeting CAR modified-UNKT cells had enhanced killing potential against MSLN positive ovarian cancer compared with the wild type UNKT, as well as MSLN-CAR-T cells. Compared with CAR-T, Single-cell microarray 32-plex proteomics revealed CAR-UNKT cells express more effector cytokines, such as perforin and granzyme B, and less interleukin-6 after activation. Moreover, our CAR-UNKT cells featured in more multifunctionality than CAR-T cells. CAR-UNKT cells also demonstrated strong antitumor activity in mouse models of ovarian cancer, with the ability to migrate and infiltrate the tumor without inducing immune memory. The fast-in and -out, enhanced and prolonged tumor killing properties of CAR-UNKT suggested a novel cure option of cellular immunotherapy in the treatment of MSLN-positive solid tumors.

Single-Cell Microarray Chip with Inverse-Tapered Wells to Maintain High Ratio of Cell Trapping.

A single-cell microarray (SCM) influenced by gravitational force is expected to be one of the simple methods in various fields such as DNA analysis and antibody production. After trapping the cells in the SCM chip, it is necessary to remove the liquid from the SCM to wash away the un-trapped cells on the chip and treat the reagents for analysis. The flow generated during this liquid exchange causes the trapped cells to drop out of conventional vertical wells. In this study, we propose an inverse-tapered well to keep trapped cells from escaping from the SCM. The wells with tapered side walls have a reduced force of flow toward the opening, which prevents trapped cells from escaping. The proposed SCM chip was fabricated using 3D photolithography and polydimethylsiloxane molding techniques. In the trapping experiment using HeLa cells, the cell residual rate increased more than two-fold for the SCM chip with the inverse-tapered well with a taper angle of 30° compared to that for the conventional vertical SCM chip after multiple rounds of liquid exchanges. The proposed well structure increases the number of trapped cells and decreases the cell dropout rate to improve the efficiency of cellular analysis.

Fabrication of single cell microarrays on a double-layered hydrogel for mitochondrial activity monitoring

Single-cell microarrays may play an important role in understanding the heterogeneity of cell populations. A hydrogel-coated micropatterned surface was prepared to capture single cells from a mixture of cells and detect their mitochondrial activity using real-time fluorescence analysis. The double-layered hydrogel micropatterns were functionalized with RGD-containing peptides using a photomask. We were able to control the number of cells immobilized on each spot of the micropattern by controlling the size of the spots. Single cells were properly arranged on the peptide-functionalized spot with aligned squares (20 μm × 20 μm), with 68% frequency of single cells. In the single-cell micropattern, a mitochondria-targetable switch-on-type of probe was employed to discriminate between the types of cells and exhibit heterogeneous cell behavior in real-time. This single cell detection technology allows for the analysis of cell function and gene expression in various environments.

Single-Cell Microarray with Microwells to Prevent Cell from Dropping Out

Single-Cell Microarray with Microwells to Prevent Cell from Dropping Out

MT-Associated Protein Dysfunction May Lead to Impaired Ppf of Megakaryocytes in Corticosteroid-Resistant/Relapse Immune Thrombocytopenia Patients

IntroductionPrimary immune thrombocytopenia (ITP) is an autoimmune disorder with reduced platelet production, and increased platelet destruction is considered the main pathogenic mechanism. However, some ITP patients present resistance to treatment with corticosteroids or relapse after a short period of remission. The existence of these patients led us to consider the possibility of other non-immune mechanisms of ITP pathogenesis. It has been reported that in some patients with persistent or chronic ITP, the pro-platelet formation of megakaryocytes (MKs) is intrinsically impaired (Rivière et al, British Journal of Haematology 2015; 170: 408). Many MT-associated proteins (MAPs) are required for megakaryopoiesis and thrombopoiesis, but the roles of MAPs in ITP patients have rarely been reported. The hypothesis of this study is that the expression and function of MAPs in corticosteroid-resistant/relapse ITP patients are abnormal and that this abnormality may lead to the impaired pro-platelet formation (PPF) and contribute to the pathogenesis in these ITP patients.MethodsThirty corticosteroid-resistant/relapse ITP patients and 10 healthy donors were enrolled in this study. MKs were isolated by cell sorting from bone marrow samples obtained by aspiration from the posterior iliac crest, and single-cell microarray and bioinformatics analysis were performed. CD34+ cells were isolated from bone marrow samples by cell sorting for in vitro culture with TPO, and on day 8, differentiated MKs were enriched. Polymerase chain reaction (PCR) and Western blot (WB) were performed to verify the expression level of MAPs in differentiated MKs. Immunofluorescence staining was performed for tubulin and MAPs and observed by inverted confocal fluorescence microscopy. In the ITP group, all trans retinoic acid (ATRA) was added to the differentiating culture process, and in the donor group, lentivirus-expressing MAP-specific siRNAs were transferred into the CD34+ cells to knock down MAP expression before culture. Proliferation, GPIIb/IIIa-GPIbIX expression, ploidy distribution and PPF were observed in the different groups. Finally, the isolated megakaryocyte progenitors (MKPs) from ITP patients and donors were transferred into irradiated NOD/SCID mice.ResultsThe bioinformatics analysis of the results showed that the expression of MAPs in corticosteroid-resistant/relapse ITP patients was relatively decreased and that the Pak2 signaling pathway may be the upstream regulator of the abnormal MAPs. The mRNA and protein levels of MAPs in cultured differentiated MKs from ITP patients were decreased compared with controls, which is consistent with the bioinformatics analysis results. The phosphorylation level of the Pak2 signaling pathway was also decreased in the ITP group. The in vitro cultured differentiated MKs from ITP patients displayed normal proliferation, GPIIb/IIIa-GPIbIX expression, and ploidy distribution, but impaired and decreased PPF compared with control groups. Immunofluorescence results of the differentiated MKs showed that the expression of MAPs and the distribution of microtubules were abnormal both in MKs and in pro-platelets from ITP patients. In the MAP-silenced donor group, the percentage of proplatelet-displaying megakaryocytes was significantly decreased. In the ITP group, when ATRA was added to the culture, MAP expression and Pak2 phosphorylation were recovered, and as a result, the PPF was increased. When transferred into the irradiated NOD/SCID mice, the platelet reconstitution of MKPs from corticosteroid-resistant/relapse ITP patients was low. This finding supported the conclusion that the abnormal MAPs in MKs could lead to decreased platelet production.ConclusionMAP expression was decreased in corticosteroid-resistant/relapse ITP patients, and this abnormality was responsible for the decreased PPF of MKs from these patients. After treatment with ATRA, the Pak2 pathway activity, the MAP expression level, and the MK function was recovered. The abnormality of the expression and function of MAPs in corticosteroid-resistant/relapse ITP patients may be a new pathogenic mechanism in these patients. DisclosuresNo relevant conflicts of interest to declare.

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics.

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Inkjet printing-assisted single-cell microarray on a hydrophobic surface chip for real-time monitoring of enzyme kinetics at single-cell level

A convenient, facile, and mask-free approach assay was developed for single-cell study by using a combination of inkjet printing technology and polydimethylsiloxane (PDMS) microchip-assisted processing. The inkjet printing technology resulted in 91% of the single-cell occupancy by individually spraying MCF-7 cells on a hydrophobic substrate and enabled the control over the number of cells with precision by strictly optimizing the printing parameters. Further, the microchip containing a cell chamber and straight channels was attached to the glass slide to explore the real-time performance of the cells. To address the performance attributes, the enzyme kinetics and various parameters of the post-printed MCF-7 cells, such as the levels of cell viability, reactive oxygen species (ROS), cell apoptosis, and proliferation, are monitored in real-time. Interestingly, high activity and proliferation, low level of ROS, and cell apoptosis demonstrated that the developed method provided a new way to the study of single-cell in-depth. Finally, ATP-induced cell proliferation of different cell number were analyzed, and the results would provide another perspective for the diagnosis and medical treatment.

Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes.

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0–20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

Aqueous Processed Biopolymer Interfaces for Single-Cell Microarrays

Single-cell microarrays are emerging tools to unravel intrinsic diversity within complex cell populations, opening up new approaches for the in-depth understanding of highly relevant diseases. However, most of the current methods for their fabrication are based on cumbersome patterning approaches, employing organic solvents and/or expensive materials. Here, we demonstrate an unprecedented green-chemistry strategy to produce single-cell capture biochips onto glass surfaces by all-aqueous inkjet printing. At first, a chitosan film is easily inkjet printed and immobilized onto hydroxyl-rich glass surfaces by electrostatic immobilization. In turn, poly(ethylene glycol) diglycidyl ether is grafted on the chitosan film to expose reactive epoxy groups and induce antifouling properties. Subsequently, microscale collagen spots are printed onto the above surface to define the attachment area for single adherent human cancer cells harvesting with high yield. The reported inkjet printing approach enables one to modulate the collagen area available for cell attachment in order to control the number of captured cells per spot, from single-cells up to double- and multiple-cell arrays. Proof-of-principle of the approach includes pharmacological treatment of single-cells by the model drug doxorubicin. The herein presented strategy for single-cell array fabrication can constitute a first step toward an innovative and environmentally friendly generation of aqueous-based inkjet-printed cellular devices.

Visualization of sequential conversion of human intermediately reprogrammed stem cells into iPS cells.

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre-iPSC lines. The iRSC-iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single-cell microarray analysis and genome editing. Here, single-cell microarray analysis showed the stage-specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E-CADHERIN as marker genes. Out of 75 OCT4-related genes, which were significantly up-regulated after the activation of OCT4, and entry into the mesenchymal-to-epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4-GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.

Computational analysis of single-cell transcriptomics data elucidates the stabilization of Oct4 expression in the E3.25 mouse preimplantation embryo

Our computational analysis focuses on the 32- to 64-cell mouse embryo transition, Embryonic day (E3.25), whose study in literature is concentrated mainly on the search for an early onset of the second cell-fate decision, the specification of the inner cell mass (ICM) to primitive endoderm (PE) and epiblast (EPI). We analysed single-cell (sc) microarray transcriptomics data from E3.25 using Hierarchical Optimal k-Means (HOkM) clustering, and identified two groups of ICM cells: a group of cells from embryos with less than 34 cells (E3.25-LNCs), and another group of cells from embryos with more than 33 cells (E3.25-HNCs), corresponding to two developmental stages. Although we found massive underlying heterogeneity in the ICM cells at E3.25-HNC with over 3,800 genes with transcriptomics bifurcation, many of which are PE and EPI markers, we showed that the E3.25-HNCs are neither PE nor EPI. Importantly, analysing the differently expressed genes between the E3.25-LNCs and E3.25-HNCs, we uncovered a non-autonomous mechanism, based on a minimal number of four inner-cell contacts in the ICM, which activates Oct4 in the preimplantation embryo. Oct4 is highly expressed but unstable at E3.25-LNC, and stabilizes at high level at E3.25-HNC, with Bsg highly expressed, and the chromatin remodelling program initialised to establish an early naïve pluripotent state. Our results indicate that the pluripotent state we found to exist in the ICM at E3.25-HNC is the in vivo counterpart of a new, very early pluripotent state. We compared the transcriptomics profile of this in vivo E3.25-HNC pluripotent state, together with the profiles of E3.25-LNC, E3.5 EPI and E4.5 EPI cells, with the profiles of all embryonic stem cells (ESCs) available in the GEO database from the same platform (over 600 microarrays). The shortest distance between the set of inner cells (E3.25, E3.5 and E4.5) and the ESCs is between the E3.25-HNC cells and 2i + LIF ESCs; thus, the developmental transition from 33 to 34 cells decreases dramatically the distance with the naïve ground state of the 2i + LIF ESCs. We validated the E3.25 events through analysis of scRNA-seq data from early and late 32-cell ICM cells.

Simultaneous Assay of Oxygen-Dependent Cytotoxicity and Genotoxicity of Anticancer Drugs on an Integrated Microchip.

Oxygen deprivation is a common feature in a variety of cancer tissues and associated with tumor progression, acquisition of antiapoptotic potential, and clinical therapeutic resistance. Thus, great interest has been aroused to develop new platforms or approaches of activity assays to impact on the hypoxic microenvironment and oxygen-dependent drug responses to improve the productivity of new drug discovery. In this study, an integrated microsystem is established to combine the cytotoxic and genotoxic tests together for continuous multiple measurements under mimicking hypoxic tumor microenvironment. We fabricated a double-layer chip device by combining a single-cell-arrayed agarose layer with a microfluidics-based oxygen gradient-generating layer using a PDMS membrane. Using tirapazamine (TPZ) and blemycin (BLM) as model anticancer drugs, we demonstrated its application and performance in single cell loading, cell cultivation, and subsequent drug treatment as well as in situ analysis of oxygen-dependent cytotoxicity and genotoxicity of anticancer drugs. The results demonstrated the opposite oxygen-dependent toxicity of TPZ and BLM, which also indicated that the formation of DNA breaks is related with cell apoptosis. Compared with the traditional assays, this device takes advantage of microfluidic phenomena to generate various oxygen concentrations while exhibiting the combinatorial diversities achieved by the single cell microarray, offering a powerful tool to study single cell behaviors and responses under different oxygen conditions with desired high-content and high-throughput capabilities.

Genomic alterations in cells involved in the atherosclerotic process

Aim: Macrophages and smooth muscle cells (SMCs) play a significant role in atherosclerosis. Genomic alteration in cells arising during an atherosclerotic process can be important for plaque instability. Our purpose was an assessment of genomic alterations in cells obtained from atherosclerotic plaque using single-cell microarray technique.

Electric-field driven assembly of live bacterial cell microarrays for rapid phenotypic assessment and cell viability testing

Microarray technology to isolate living cells using external fields is a facile way to do phenotypic analysis at the cellular level. We have used alternating current dielectrophoresis (AC-DEP) to drive the assembly of live pathogenic Salmonella typhi (S.typhi) and Escherichia coli (E.coli) bacteria into miniaturized single cell microarrays. The effects of voltage and frequency were optimized to identify the conditions for maximum cell capture which gave an entrapment efficiency of 90% in 60 min. The chip was used for calibration-free estimation of cellular loads in binary mixtures and further applied for rapid and enhanced testing of cell viability in the presence of drug via impedance spectroscopy. Our results using a model antimicrobial sushi peptide showed that the cell viability could be tested down to 5 μg/mL drug concentration under an hour, thus establishing the utility of our system for ultrafast and sensitive detection.

Single Cell Microarrays Fabricated by Microscale Plasma-Initiated Protein Patterning (μPIPP).

Micropatterned arrays considerably advanced single cell fluorescence time-lapse measurements by providing standardized boundary conditions for thousands of cells in parallel. In these assays, cells are forced to adhere to defined microstructured protein islands separated by passivated, nonadhesive areas. Here we provide a detailed protocol on how to reproducibly fabricate high quality single cell arrays by microscale plasma-initiated protein patterning (μPIPP). Advantages of μPIPP arrays are the ease of preparation and the unrestricted choice of substrates as well as proteins. We demonstrate how the arrays enable the efficient measurement of single cell time trajectories using automated data acquisition and data analysis by example of single cell gene expression after mRNA transfection and time courses of single cell apoptosis. We discuss the more general use of the protocol for assessment of single cell dynamics with the help of fluorescent reporters.

Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31–40 µm upper diameter, 11–20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

Fast Single-Cell Patterning for Study of Drug-Induced Phenotypic Alterations of HeLa Cells Using Time-of-Flight Secondary Ion Mass Spectrometry.

A facile single-cell patterning (ScP) method was developed and integrated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) for the study of drug-induced cellular phenotypic alterations. Micropatterned poly(dimethylsiloxane) (PDMS) stencil film and centrifugation-assisted cell trapping were combined for the preparation of on-surface single-cell microarrays, which exhibited both high site occupancy (>90%) and single-cell resolution (>97%). TOF-SIMS is a surface-sensitive mass spectrometry and is increasingly utilized in biological studies. Here we demonstrated, for the first time, its successful application in high-throughput single-cell analysis. Drug-induced phenotypic alterations of HeLa cells in the early stage of apoptosis were investigated using TOF-SIMS. The major molecular sources of variations were analyzed by principle component analysis (PCA).

Differential network analyses of Alzheimer's disease identify early events in Alzheimer's disease pathology.

In late-onset Alzheimer's disease (AD), multiple brain regions are not affected simultaneously. Comparing the gene expression of the affected regions to identify the differences in the biological processes perturbed can lead to greater insight into AD pathogenesis and early characteristics. We identified differentially expressed (DE) genes from single cell microarray data of four AD affected brain regions: entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC), and middle temporal gyrus (MTG). We organized the DE genes in the four brain regions into region-specific gene coexpression networks. Differential neighborhood analyses in the coexpression networks were performed to identify genes with low topological overlap (TO) of their direct neighbors. The low TO genes were used to characterize the biological differences between two regions. Our analyses show that increased oxidative stress, along with alterations in lipid metabolism in neurons, may be some of the very early events occurring in AD pathology. Cellular defense mechanisms try to intervene but fail, finally resulting in AD pathology as the disease progresses. Furthermore, disease annotation of the low TO genes in two independent protein interaction networks has resulted in association between cancer, diabetes, renal diseases, and cardiovascular diseases.

GnRH neuron type-specific transcriptome analysis by laser captured single-cell microarray in the medaka

Most vertebrates possess at least two gonadotropin-releasing hormone (GnRH) neuron types. To understand the physiological significance of the multiple GnRH systems in the brain, we examined three GnRH neuron type-specific transcriptomes using single-cell microarray analyses in the medaka (Oryzias latipes). A microarray profile of the three GnRH neuron types revealed five genes that are uniquely expressed in specific GnRH neuron types. GnRH1 neurons expressed three genes that are homologous to functionally characterised genes, GnRH2 neurons uniquely expressed one unnamed gene, and GnRH3 neurons uniquely expressed one known gene. These genes may be involved in the modulation or maintenance of each GnRH neuron type.

Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.