Single-cell Western Blot Research Articles

Microparticle Delivery of Protein Markers for Single-Cell Western Blotting from Microwells.

Immunoblotting confers protein identification specificity beyond that of immunoassays by prepending protein electrophoresis (sizing) to immunoprobing. To accurately size protein targets, sample analysis includes concurrent analysis of protein markers with known molecular masses. To incorporate protein markers in single-cell western blotting, microwells are used to isolate individual cells and protein marker-coated microparticles. A magnetic field directs protein-coated microparticles to >75% of microwells, so as to 1) deliver a quantum of protein marker to each cell-laden microwell and 2) synchronize protein marker solubilization with cell lysis. Nickel-coated microparticles are designed, fabricated, and characterized, each conjugated with a mixture of histidine-tagged proteins (42.3-100 kDa). Imidazole in the cell lysis buffer solubilizes protein markers during a 30 s cell lysis step, with an observed protein marker release half-life of 4.46 s. Across hundreds of individual microwells and different microdevices, robust log-linear regression fits (R2 > 0.97) of protein molecular mass and electrophoretic mobility are observed. The protein marker and microparticle system is applied to determine the molecular masses of five endogenous proteins in breast cancer cells (GAPDH, β-TUB, CK8, STAT3, ER-α), with <20% mass error. Microparticle-delivered protein standards underpin robust, reproducible electrophoretic cytometry that complements single-cell genomics and transcriptomics.

Electrophoretic cytopathology resolves ERBB2 forms with single-cell resolution

In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies. Our microfluidic, single-cell western blot, employs electrophoretic separations to resolve full-length HER2 from the smaller t-erbB2 in each ~28 pL single-cell lysate. Subsequently, a pan-HER2 antibody detects all resolved HER2 protein forms via immunoprobing. In analysis of eight breast tumor biopsies, we identified two tumors comprised of 15% and 40% t-erbB2-expressing cells. By single-cell western blotting of the t-erbB2-expressing cells, we observed statistically different ratios of t-erbB2 proteins to full-length HER2 expression. Further, target multiplexing and clustering analyses scrutinized signaling, including ribosomal S6, within the t-erbB2-expressing cell subpopulation. Taken together, cytometric assays that report both protein isoform profiles and signaling state offer cancer classification taxonomies with unique relevance to precisely describing drug resistance mechanisms in which oncoprotein isoforms/fragments are implicated.

Single-cell protein analysis in support of biomarker evaluation for breast cancer immunotherapeutics.

10 Background: Triple-negative breast cancer (TNBC) is being tested for PD-1/PD-L1 checkpoint inhibitors combination therapy. The relationship between tumor expression of PD-L1 and patient outcomes has been established using immunohistochemistry (IHC) biomarker assays across various malignancies. PD-L1 is expressed in tumor and immune cells within the tumors in TNBC, but only immune-cell PD-L1 is associated with response to anti-PD-L1 (atezolizumab) so differentiating tumor vs. immune PD-L1 expression is essential. The currently available IHC biomarker assays that relay on tissue biopsies struggle to provide clinically meaningful identification of responders vs. non-responders. Poor sensitivity and specificity is partially attributed to tumor heterogeneity that is not well-represented in these biopsies. In addition, PD-L1 expression may vary over time due to disease progression or treatment. Since tissue biopsy collection is often an invasive procedure, it is restricted. Needle biopsies can mitigate some of these issues as a minimally invasive method to probe multiple regions within several cancer lesions across several time points. MILO, a new single cell Western Blot (WB) technology allows quantitative measurements of multiple protein biomarkers within one cell in small samples. Methods: To evaluate the capability of MILO to differentiate between PD-L1 in immune and tumor cell populations, we interrogated BC cell lines as well as peripheral blood mononuclear cells (PBMCs) mixed in known ratios. We used MDA-MB-231 that shows high PD-L1 and low HER2 expression and BT-474, which shows the inverse expression profile for these biomarkers along with PBMCs as PD-L1+ immune cells. Primary breast tumor tissues are currently interrogated to establish utility in needle biopsies. Results: MILO demonstrated heterogeneity at the single-cell level of HER2 and PD-L1 expression in BC tumor cells and enabled differentiation between PD-L1 expression in immune vs. tumor cells using cell-specific markers and low cell numbers. Conclusions: MILO can improve patient selection and prediction of response to immune checkpoint inhibition by promoting the utility of needle biopsies to complement IHC of core biopsies.

Single Cell Proteomics for Molecular Targets in Lung Cancer: High-Dimensional Data Acquisition and Analysis.

In the proteomic and genomic era, lung cancer researchers are increasingly under challenge with traditional protein analyzing tools. High output, multiplexed analytical procedures are in demand for disclosing the post-translational modification, molecular interactions and signaling pathways of proteins precisely, specifically, dynamically and systematically, as well as for identifying novel proteins and their functions. This could be better realized by single-cell proteomic methods than conventional proteomic methods. Using single-cell proteomic tools including flow cytometry, mass cytometry, microfluidics and chip technologies, chemical cytometry, single-cell western blotting, the quantity and functions of proteins are analyzed simultaneously. Aside from deciphering disease mechanisms, single-cell proteomic techniques facilitate the identification and screening of biomarkers, molecular targets and promising compounds as well. This review summarized single-cell proteomic tools and their use in lung cancer.

BioSpotlight / Citations

BioTechniquesVol. 62, No. 4 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114530AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Stabler expression in stable cell linesA major issue with stable cell lines generated by standard methods is the heterogeneous levels of recombinant protein expression in different cell clones due to the integration of variable numbers of the transgene and/or position effects caused by integration of the transgene into random locations in the genome. In addition, the use of strong heterologous promoters in transgenic vectors can drive expression to very high, non-physiological levels that harm the cells. In this month's issue, researchers led by Brian Chen at McGill University describe an efficient method for stable cell line generation that achieves controlled, physiological expression of a transgene. Using CRISPR/Cas9 gene editing, the team inserted a single copy of a transgene at a specific gene locus, placing it under the control of an endogenous promoter with the desired level of gene expression. Their transgenic vector also includes a protein quantitation reporter gene to produce an equimolar amount of a fluorescent protein, allowing easy quantitation of the recombinant protein.See “Generating stable cell lines with quantifiable protein production using CRISPR/Cas9 mediated knock-in” on page 165.Two colors are better than one for dna methylation assayThe cytosine extension assay (CEA) assesses global DNA methylation levels by tracking the methylation of a CpG-containing restriction enzyme site, for example CCGG, which when methylated cannot be digested by HpaII but can be digested by its isoschizomer, MspI. The relative digestion of CCGG sites in a DNA sample by HpaII compared to MspI reflects the extent of DNA methylation and is assessed in CEA by the incorporation of a fluorescently labeled cytosine through end-filling by DNA polymerase of the 5′ guanosine overhang at the HpaII/MspI cut site. In this issue of BioTechniques, Craig Parfett and his colleagues at Health Canada present an improved version of CEA. In standard CEA, the same fluorescently colored cytosine is used in the extension reactions following HpaII and MspI digestion. In their version, Parfett's team used a different fluorescently colored cytosine for the extension reaction of each digest, after which the reactions were combined for downstream purification and signal detection. This two-color CEA showed greater precision compared to the one-color CEA and is well-suited to high-throughput screening.See “Two-color fluorescent cytosine extension assay for the determination of global DNA methylation” on page 157.Root problemsPlant biologists have long been interested in understanding the interactions that occur in nature between microorganisms and plant roots. Plants both obtain information about their environment and send out signals to microbes through their roots. However, deciphering the interplay between plant roots and the microbial communities surrounding them has proven difficult. Now, researchers report in PNAS a new microfluidic-based device designed specifically for monitoring root–bacteria interactions over time. The device, which the authors named the tracking root interaction system (TRIS), is composed of 9 independent microfluidic chambers where roots can be established and into which bacteria are introduced for monitoring. The chambers are coupled to a microscope assembly that makes it possible to obtain both spatial and temporal information on root–bacteria interactions. In proof-of-concept experiments, the authors fluorescently labeled bacteria and were able to observe root colonization in real time over extended periods using their TRIS device.Massalha, H. et al. 2017. Live imaging of root-bacteria interactions in a microfluidics setup. Proc Natl Acad Sci USA. doi:10.1073/pnas.1618584114.Western blotting goes smallWestern blotting is a commonly used technique for studying cellular proteins. While effective when using large numbers of cells as starting material, western blot analysis has proven more difficult when scaled down for studies of single cells with their much lower protein concentrations. Seeking to overcome this limitation and extend the range of protein analysis, researchers describe a new rare-cell, single-cell western blotting procedure in Nature Communications. Building on previous work that established the feasibility of single-cell resolution western blotting, the authors sought to measure panels of proteins from extremely rare circulating tumor cell (CTC) isolates. For protein analysis, each CTC was placed into an individual microwell, where the cell was first lysed and then immediately electrophoresed into a polyacrylamide matrix. Following a miniaturized version of polyacrylamide gel electrophoresis (PAGE), in-gel immunoprobing was done on the size-separated CTC proteins. Using this method, the authors were able to successfully perform multiplexed protein analyses and report protein expression on a per-cell basis.Sinkala, E. et al. 2017. Profiling protein expression in circulating tumor cells using microfluidic western blotting. Nat Commun. 8:14622.FiguresReferencesRelatedDetails Vol. 62, No. 4 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print April 2017 Information© 2017 Author(s)PDF download

Subcellular western blotting of single cells

Although immunoassays are the de facto standard for determining subcellular protein localization in individual cells, antibody probe cross-reactivity and fixation artifacts remain confounding factors. To enhance selectivity while providing single-cell resolution, we introduce a subcellular western blotting technique capable of separately assaying proteins in the 14 pL cytoplasm and 2 pL nucleus of individual cells. To confer precision fluidic control, we describe a passive multilayer microdevice that leverages the rapid transport times afforded by miniaturization. After isolating single cells in microwells, we apply single-cell differential detergent fractionation to lyse and western blot the cytoplasmic lysate, whereas the nucleus remains intact in the microwell. Subsequently, we lyse the intact nucleus and western blot the nuclear lysate. To index each protein analysis to the originating subcellular compartment, we utilize bi-directional electrophoresis, a multidimensional separation that assays the lysate from each compartment in a distinct region of the separation axis. Single-cell bi-directional electrophoresis eliminates the need for semi-subjective image segmentation algorithms required in immunocytochemistry. The subcellular, single-cell western blot is demonstrated for six targets per cell, and successfully localizes spliceosome-associated proteins solubilized from large protein and RNA complexes, even for closely sized proteins (a 7 kDa difference). Measurement of NF-κB translocation dynamics in unfixed cells at 15-min intervals demonstrates reduced technical variance compared with immunofluorescence. This chemical cytometry assay directly measures the nucleocytoplasmic protein distribution in individual unfixed cells, thus providing insight into protein signaling in heterogeneous cell populations.

Single cell-resolution western blotting.

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4-6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

Abstract 352: HER2 protein isoform heterogeneity investigated by single-cell western blotting

Abstract Accounting for ∼20% of breast cancers, half of metastatic HER2-positive breast cancer patients treated with trastuzumab plus chemotherapy will experience progression of disease within one year. Trastuzumab treatment, though associated with improvement in median overall survival, still sees drug resistance as an unmet challenge. Differential response to therapy may arise from tumor heterogeneity. Further complicating the situation is the fact that HER2/erbB2 biology is complex. HER2 is not simply one full-length protein (p185) overexpressed on tumor cell surfaces, but also includes truncated isoforms, t-erbB2 (e.g., 95 and 110 kDa isoforms) lacking the extracellular domain targeted by trastuzumab (and pertuzumab and T-DM1). The t-erbB2 isoforms are implicated in tumorigenesis, metastasis, and therapeutic response. Given existing pathology and life sciences tools, the high heterogeneity and need for single cell resolution presents a tremendous challenge. We introduce here a novel microfluidic device to quantitatively assess HER2 protein isoforms in heterogeneous tumor biopsies with single-cell resolution. The single-cell western blot (scWB) provides both the sensitivity and specificity needed to detect t-erbB2 isoforms owing to use of a single-cell electrophoretic protein separation followed by immunoprobing. The assay further provides enough sensitivity to obviate the need for cell pooling, which obscures cell-to-cell heterogeneity. We assay ∼1000's of single cells on one microscope slide in a 4-6 hr workflow. First, we validated that the scWB can separate full length (p185) from truncated (p95) HER2 in single p185- and p95-genetically engineered CHO cells. The p95 was resolved from p185 protein in a short ∼1 mm separation lane (resolution = 0.97±0.03, n&amp;gt;500 cells). Next, we assessed a HER2-positive breast cancer cell line (BT474) and found &amp;lt;3% t-erbB2 expressing cells. To further investigate the effect of t-erbB2 in HER2-related signaling, we examined phospho-proteins including p-HER2, p-HER3, p-ERK Thr187/Tyr185, p-ERK Thr202/Tyr204, p-rs6 from each t-erbB2 expressing BT474 cell. Protein activation in t-erbB2 expressing cells was similar to cells expressing full length HER2. This first-in-kind study demonstrates a powerful single-cell resolution multiplexing capability including for phospho-proteins. We are now applying the novel scWB to measure t-erbB2 in HER2-positive and triple positive (ER/PR/HER2) breast tumor biopsies and will report our results at AACR. Taken together, the microfluidic single-cell resolution western provides a sensitive, quantitative, and multiplexed tool to assay clinically-relevant oncoprotein isoforms that are currently difficult or impossible to assay. Correlating isoform expression ratios with clinical outcomes may confer a more comprehensive understanding of drug resistance responses derived from oncoprotein isoform profiles within heterogeneous cancer cell populations. Citation Format: Ginny (Chi-Chih) Kang, Toby M. Ward, Jessica Bockhorn, Mark D. Pegram, Amy E. Herr. HER2 protein isoform heterogeneity investigated by single-cell western blotting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 352.

Abstract 1997: Label-free concentration of viable breast cancer CTCs for single cell Western blotting

Abstract The isolation of circulating tumor cells (CTCs) in blood can aid in cancer prognosis, characterize genetic mutations for targeted therapies, and elucidate the biological mechanisms of metastasis. While CTCs provide clinical utility, there are still several technical difficulties with CTC isolation and analysis. CTCs are very rare (1-10 cells/mL of blood) and current isolation methods rely on immunocapture, require long sample preparation, and fail to deliver viable cells. Further, recent advances in single cell analysis of CTCs have been limited to genomic studies, in which DNA/mRNA does not provide a complete picture of CTCs at the functional level, as mRNA expression may only account for a portion of protein expression. The ability to study and measure the protein expression of CTCs could provide insight into the mechanisms driving cancer progression and signaling dysregulation. Unfortunately, conventional proteomic tools (e.g. immunocytochemistry and Western blot) either do not provide sufficient specificity or sensitivity toward single cell levels. Thus, there is a lack of tools that can provide quantitative protein analysis for rare CTCs. To address this gap, we enable rare cell Western blotting by combining isolation and proteomic technologies in a unique workflow: (i) Label-free Vortex enrichment of rare larger cells from blood samples. Larger cancer cells are trapped within an array of microscale fluid vortices that form in simple rectangular reservoirs, while smaller blood cells pass through. The cancer cells are stably maintained in the vortices, allowing for solution exchange, followed by release upon lowering the flow rate. With this device, we were able to release a small volume (∼500 μL) of concentrated, viable cancer cells (MCF7) from healthy blood, at high throughput, capture efficiency (35%), purity (&amp;gt;80%), and ∼10,000 fold enrichment. CTCs were also successfully extracted and enumerated from the blood of patients with breast cancer (N = 18, from 0.75 to 23.25 CTCs/mL). (ii) High-specificity and multiplexed protein analysis of rare cancer cells with single cell Western blotting (scWB). Enriched CTCs are placed into microwells patterned into a polyacrylamide gel for the scWB assay (in-situ chemical lysis in the microwells, electrophoresis into bulk gel layer, photo-immobilization, and subsequent antibody-probing of immobilized proteins). Here, we demonstrate scWB compatibility with rare cells by spiking MCF7s into normal blood and performing protein separations. The scWB is multiplex and can assay several target proteins simultaneously. Cells stained for surface markers (anti-EpCAM) are also compatible, and the scWB can resolve complete immunocomplexes without disrupting the location of the intracellular targets (GAPDH, ERK). With the integration of these technologies, we push the limits of quantitative protein analysis of CTCs, an essential step to better understand these rare populations. Citation Format: Elly Sinkala, Elodie Sollier, Corinne Renier, James Che, Stefanie S. Jeffrey, Amy E. Herr. Label-free concentration of viable breast cancer CTCs for single cell Western blotting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1997. doi:10.1158/1538-7445.AM2015-1997

Abstract 4919: Tumor heterogeneity represented in human cancer cell lines

Abstract EGFR has been found frequently mutated in human cancers, and more than 10% lung cancer patients associated with EGFR mutation. In addition, several critical mutations within the EGFR have been shown as gain of function mutations or can cause drug resistance to the EGFR inhibitors. A panel of ATCC tumor cell lines has been characterized for EGFR mutation and EGFR signaling pathway. Particularly, lung cell line NCI-H1975, which contains both drug sensitive mutation EGFR p.L858R and drug resistant mutation p.T790M, has displayed a resistance to EGFR inhibitor.Overall, NCI-H1975 cell line can be used as a nice model to study drug resistance, as well as to screen novel EGFR inhibitors that overcome the p.T790M-mediated resistance. Moreover, we have used next generation sequencing to analyze the mutation allelic frequency of both mutations, and observed tumor heterogeneity within this cell line. Impact of cell passage numbers on the mutation allele frequency of the cell line was also examined. Furthermore, a novel single cell western blot assay has been used to analyze the EGFR mutation, EGFR protein expression and downstream signaling within individual cells of three lung cancer cell lines at the single cell level. Collectively, NCI-H1975 cell line represents the acquired drug resistance found in clinical cancer patients, and this study demonstrates the tumor heterogeneity represented in human cell lines. Citation Format: Lysa-Anne Volpe, John Foulke, Michael Jackson, Luping Chen, David H. Randle, Hannah J. Gitschier, Kelly Gardner, Fang Tian. Tumor heterogeneity represented in human cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2015-4919

Single-cell western blotting.

Cell heterogeneity is a variation in cellular processes in functionally similar cells. Cells from the same tissue which are considered genetically identical may have difference in size, structure, and level of protein expression which can lead to major impact on the functions of cell leading to difference in physiological consequences. Single-cell proteome-wide studies are used to detect cell heterogeneity. Flow cytometry and immunocytochemistry do play an important role in evaluating cell heterogeneity. However, these methods are based on separation by antibodies with limited specificity. Cross-reactivity can occur leading to bias in result. Western blot is done to separate the proteins according to molecular weight. Therefore, off-target and on-target signals can be discriminated. Detection of protein expression from a tissue can be done with the help of western blot. However, it is unable to differentiate protein expression of individual cells. For detection of this cell-to-cell variation, a highly advanced technique termed "single-cell western blotting" is carried out. Single-cell western blot has enabled us to detect protein expression at cellular level at a fairly advanced high resolution using a western blot designed to assess cell heterogeneity.

Single-Cell Western Blotting.

Little headway has been made in single cell protein analysis, aside from tools that rely solely on antibody-probe based detection (i.e., flow cytometry, immunocytochemistry), which are limited by low specificity and multiplexing capabilities. To address these protein analysis gaps, we have introduced a single-cell western blot (scWestern). The protein assay is capable of highly specific analysis by coupling antibody-based detection with a polyacrylamide gel electrophoresis (PAGE) protein separation. Cells are settled via gravity into polyacrylamide (PA) microwells, chemically lysed in the wells, and then subjected to PAGE through the walls of the microwells and into the surrounding PA gel. Over a thousand single-cell separations are performed simultaneously, and multiple protein targets of interest are investigated. After PAGE separation, photo-immobilization of all proteins to the gel allows for antibody probing and lends to the archival quality of the scWestern assay where new proteins targets can be investigated months after the initial separations are performed.

Single-cell Western blotting after whole-cell imaging to assess cancer chemotherapeutic response.

Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.

Research highlights: microfluidic single-cell analysis from nucleic acids to proteins to functions

We highlight recent reports using microfluidic systems to perform single-cell analysis. It has been demonstrated on numerous occasions that population averages are often not representative of single-cell behavior. These differences in behavior can be caused by stochastic fluctuations in temporal response, changes in the surrounding instructive environment, or hard-coded genetic changes. Because of the similar length scales, microfluidic approaches have been well-suited to isolating, analyzing, and culturing single-cells to better understand this heterogeneity. Here, we discuss recent works in which microfluidic researchers have extended single-cell characterization approaches, in order to improve analysis from nucleic acids to proteins to final functional behavior. Nucleic acid detection can be amplified beyond what is possible with fluorescence in situ hybridization using droplet-enabled PCR-activated cell sorting. Multiplexed protein detection that overcomes the problems with off-target antibody binding which are associated with traditional immunofluorescence is achieved with single-cell western blotting. Proliferation, migration, and secretion are analyzed in rare circulating tumor cells isolated in microwells. The next steps will be getting these new tools into the hands of a growing number of biologists and developing new tools to report on single-cell epigenetic modifications.

Single-cell western blotting

Single-cell western blotting

Single-cell western blotting.

To measure cell-to-cell variation in protein-mediated functions — a hallmark of biological processes — we developed an approach to conduct ~103 concurrent single-cell western blots (scWesterns) in ~4 hours. A microscope slide supporting a 30 µm-thick photoactive polyacrylamide gel enables western blotting comprised of: settling of single cells into microwells, lysis in situ, gel electrophoresis, photoinitiated blotting to immobilize proteins, and antibody probing. We apply this scWestern to monitor single rat neural stem cell differentiation and responses to mitogen stimulation. The scWestern quantifies target proteins even with off-target antibody binding, multiplexes to 11 protein targets per single cell with detection thresholds of <30,000 molecules, and supports analyses of low starting cell numbers (~200) when integrated with fluorescence activated cell sorting. The scWestern thus overcomes limitations in single-cell protein analysis (i.e., antibody fidelity, sensitivity, and starting cell number) and constitutes a versatile tool for the study of complex cell populations at single-cell resolution.

A novel ruthenium(II)–polypyridyl complex inhibits cell proliferation and induces cell apoptosis by impairing DNA damage repair

Ruthenium complexes are widely recognized as one of the most promising DNA damaging chemotherapeutic drugs. The main goal of this study was to explore the anticancer activity and underlying mechanisms of [Ru(phen)2(p-BrPIP)](ClO4)2, a novel chemically synthesized ruthenium (Ru) complex. To this end, we employed MTT assays to determine the anticancer activity of the complex, and performed single-cell gel electrophoresis (SCGE) and Western blotting to evaluate DNA damage. Our results showed that the Ru(II)–poly complex caused severe DNA damage, possibly by downregulating key factors involved in DNA repair pathways, such as proliferating cell nuclear antigen (PCNA) and ring finger protein 8 (RNF8). In addition, this complex induced cell apoptosis by upregulating both p21 and p53. Taken together, our findings demonstrate that the Ru(II)–poly complex exhibits antitumour activity by inducing cell apoptosis, which results from the accumulation of large amounts of unrepaired DNA damage.