Applying single-cell highly multiplexed secretome proteomics to characterize immunotherapeutic products and predict clinical responses

Genetically and phenotypically identical immune cell populations can be highly heterogenous in terms of their immune functions and protein secretion profiles. The microfluidic chip-based single-cell highly multiplexed secretome proteomics enables characterization of cellular heterogeneity of immune responses at different cellular and molecular layers. Increasing evidence has demonstrated that polyfunctional T cells that simultaneously produce 2+ proteins per cell at the single-cell level are key effector cells that contribute to the development of potent and durable cellular immunity against pathogens and cancers. The functional proteomic technology offers a wide spectrum of cellular function assessment and can uniquely define highly polyfunctional cell subsets with cytokine signatures from live individual cells. This high-dimensional single-cell analysis provides deep dissection into functional heterogeneity and helps identify predictive biomarkers and potential correlates that are crucial for immunotherapeutic product design optimization and personalized immunotherapy development to achieve better clinical outcomes.     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Advanced Cell Mapping Visualizations for Single Cell Functional Proteomics Enabling Patient Stratification

Highly multiplexed single‐cell functional proteomics has emerged as one of the next‐generation toolkits for a deeper understanding of functional heterogeneity in cell. Different from the conventional population‐based bulk and single‐cell RNA‐Seq assays, the microchip‐based proteomics at the single‐cell resolution enables a unique identification of highly polyfunctional cell subsets that co‐secrete many proteins from live single cells and most importantly correlate with patient response to a therapy. The 32‐plex IsoCode chip technology has defined a polyfunctional strength index (PSI) of pre‐infusion anti‐CD19 chimeric antigen receptor (CAR)‐T products, that is significantly associated with patient response to the CAR‐T cell therapy. To complement the clinical relevance of the PSI, a comprehensive visualization toolkit of 3D uniform manifold approximation and projection (UMAP) and t‐distributed stochastic neighbor embedding (t‐SNE) in a proteomic analysis pipeline is developed, providing more advanced analytical algorithms for more intuitive data visualizations. The UMAP and t‐SNE of anti‐CD19 CAR‐T products reveal distinct cytokine profiles between nonresponders and responders and demonstrate a marked upregulation of antitumor‐associated cytokine signatures in CAR‐T cells from responding patients. Using this powerful while user‐friendly analytical tool, the multi‐dimensional single‐cell data can be dissected from complex immune responses and uncover underlying mechanisms, which can promote correlative biomarker discovery, improved bioprocessing, and personalized treatment development.     

Design of recombinant antibody microarrays for cell surface membrane proteomics

Generating proteomic maps of membrane proteins, common targets for therapeutic interventions and disease diagnostics, has turned out to be a major challenge. Antibody-based microarrays are among the novel rapidly evolving proteomic technologies that may enable global proteome analysis to be performed. Here, we have designed the first generation of a scaleable human recombinant scFv antibody microarray technology platform for cell surface membrane proteomics as well as glycomics targeting intact cells. The results showed that rapid and multiplexed profiling of the cell surface proteome (and glycome) could be performed in a highly specific and sensitive manner and that differential expression patterns due to external stimuli could be monitored.     

A high‐throughput all‐optical laser‐scanning imaging flow cytometer with biomolecular specificity and subcellular resolution

Image‐based cellular assay advances approaches to dissect complex cellular characteristics through direct visualization of cellular functional structures. However, available technologies face a common challenge, especially when it comes to the unmet need for unraveling population heterogeneity at single‐cell precision: higher imaging resolution (and thus content) comes at the expense of lower throughput, or vice versa. To overcome this challenge, a new type of imaging flow cytometer based upon an all‐optical ultrafast laser‐scanning imaging technique, called free‐space angular‐chirp‐enhanced delay (FACED) is reported. It enables an imaging throughput (>20 000 cells s^?1) 1 to 2 orders of magnitude higher than the camera‐based imaging flow cytometers. It also has 2 critical advantages over optical time‐stretch imaging flow cytometry, which achieves a similar throughput: (1) it is widely compatible to the repertoire of biochemical contrast agents, favoring biomolecular‐specific cellular assay and (2) it enables high‐throughput visualization of functional morphology of individual cells with subcellular resolution. These capabilities enable multiparametric single‐cell image analysis which reveals cellular heterogeneity, for example, in the cell‐death processes demonstrated in this work—the information generally masked in non‐imaging flow cytometry. Therefore, this platform empowers not only efficient large‐scale single‐cell measurements, but also detailed mechanistic analysis of complex cellular processes.      

A novel encoded particle technology that enables simultaneous interrogation of multiple cell types

The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel ways.     

Proteomic differences in recombinant CHO cells producing two similar antibody fragments

Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the production of biopharmaceuticals. To overcome unfavorable features of CHO cells, a lot of effort is put into cell engineering to improve phenotype. “Omics” studies investigating elevated growth rate and specific productivities as well as extracellular stimulus have already revealed many interesting engineering targets. However, it remains largely unknown how physicochemical properties of the recombinant product itself influence the host cell. In this study, we used quantitative label‐free LC‐MS proteomic analyses to investigate product‐specific proteome differences in CHO cells producing two similar antibody fragments. We established recombinant CHO cells producing the two antibodies, 3D6 and 2F5, both as single‐chain Fv‐Fc homodimeric antibody fragments (scFv‐Fc). We applied three different vector strategies for transgene delivery (i.e., plasmid, bacterial artificial chromosome, recombinase‐mediated cassette exchange), selected two best performing clones from transgene variants and transgene delivery methods and investigated three consecutively passaged cell samples by label‐free proteomic analysis. LC‐MS‐MS profiles were compared in several sample combinations to gain insights into different aspects of proteomic changes caused by overexpression of two different heterologous proteins. This study suggests that not only the levels of specific product secretion but the product itself has a large impact on the proteome of the cell. Biotechnol. Bioeng. 2016;113: 1902–1912. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels

The cytokine TGFβ provides important information during embryonic development, adult tissue homeostasis, and regeneration. Alterations in the cellular response to TGFβ are involved in severe human diseases. To understand how cells encode the extracellular input and transmit its information to elicit appropriate responses, we acquired quantitative time‐resolved measurements of pathway activation at the single‐cell level. We established dynamic time warping to quantitatively compare signaling dynamics of thousands of individual cells and described heterogeneous single‐cell responses by mathematical modeling. Our combined experimental and theoretical study revealed that the response to a given dose of TGFβ is determined cell specifically by the levels of defined signaling proteins. This heterogeneity in signaling protein expression leads to decomposition of cells into classes with qualitatively distinct signaling dynamics and phenotypic outcome. Negative feedback regulators promote heterogeneous signaling, as a SMAD7 knock‐out specifically affected the signal duration in a subpopulation of cells. Taken together, we propose a quantitative framework that allows predicting and testing sources of cellular signaling heterogeneity.     

Multi-Q: a fully automated tool for multiplexed protein quantitation

The iTRAQ labeling method combined with shotgun proteomic techniques represents a new dimension in multiplexed quantitation for relative protein expression measurement in different cell states. To expedite the analysis of vast amounts of spectral data, we present a fully automated software package, called Multi-Q, for multiplexed iTRAQ-based quantitation in protein profiling. Multi-Q is designed as a generic platform that can accommodate various input data formats from search engines and mass spectrometer manufacturers. To calculate peptide ratios, the software automatically processes iTRAQ's signature peaks, including peak detection, background subtraction, isotope correction, and normalization to remove systematic errors. Furthermore, Multi-Q allows users to define their own data-filtering thresholds based on semiempirical values or statistical models so that the computed results of fold changes in peptide ratios are statistically significant. This feature facilitates the use of Multi-Q with various instrument types with different dynamic ranges, which is an important aspect of iTRAQ analysis. The performance of Multi-Q is evaluated with a mixture of 10 standard proteins and human Jurkat T cells. The results are consistent with expected protein ratios and thus demonstrate the high accuracy, full automation, and high-throughput capability of Multi-Q as a large-scale quantitation proteomics tool. These features allow rapid interpretation of output from large proteomic datasets without the need for manual validation. Executable Multi-Q files are available on Windows platform at http://ms.iis.sinica.edu.tw/Multi-Q/.     

A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (～1 × 10(4) cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen-specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen-specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.     

Integrated single‐cell analysis shows Pichia pastoris secretes protein stochastically

The production of heterologous proteins by secretion from cellular hosts is an important determinant for the cost of biotherapeutics. A single‐cell analytical method called microengraving was used to examine the heterogeneity in secretion by the methylotrophic yeast Pichia pastoris. We show that constitutive secretion of a human Fc fragment by P. pastoris is not cell‐cycle dependent, but rather fluctuates between states of high and low productivity in a stochastic manner. Biotechnol. Bioeng. 2010;106: 319–325. © 2010 Wiley Periodicals, Inc.     

Microdissection—An Essential Prerequisite for Spatial Cancer Omics

The problem with cancer tissue is that its intratumoral heterogeneity and its complexity is extremely high as cells possess, depending on their location and function, different mutations, different mRNA expression and the highest intricacy in the protein pattern. Prior to genomic and proteomic analyses, it is therefore indispensable to identify the exact part of the tissue or even the exact cell. Laser‐based microdissection is a tried and tested technique able to produce pure and well‐defined cell material for further analysis with proteomic and genomic techniques. It sheds light on the heterogeneity of cancer or other complex diseases and enables the identification of biomarkers. This review aims to raise awareness for the reconsideration of laser‐based microdissection and seeks to present current state‐of‐the‐art combinations with omic techniques.     

Simultaneous detection of DNA synthesis, activation and cytokine secretion in collagen II (250-270)-activated T lymphocytes by flow cytometry

T cell activation and secretion of cytokines from activated peripheral blood mononuclear cells (PBMC) in culture have traditionally been measured by 3H-thymidine incorporation for assessment of cell proliferation. However, this method has many disadvantages that limit its usage in analyzing antigen-specific T responses, because of the low specific frequencies of the cells. Collagen II (250-270) may be an important autoantigen involved in the pathology of rheumatoid arthritis (RA). To further study the specific T cells response to CII 250-270, we developed an improved method for measuring lymphocyte proliferation and activation, and intracellular cytokine production, by flow cytometry at the single cell level. BrdU, an analog of thymidine, was incorporated into cellular DNA as a marker of individual cell proliferation. The cells were fixed and permeabilized, and a monoclonal antibody against BrdU conjugated with a fluorescent dye was used to measure BrdU incorporation. A Tris staining technique for the simultaneous determination of cell surface activation markers (CD69 or CD25) and intracellular cytokine production was also used and the parameters were assessed by 3-color flow cytometry. Optimal conditions were selected to improve the sensitivity and specificity of the assays. This method allowed simultaneous detection of lymphocytic DNA synthesis, phenotype analysis and cytokine production at the single cell level, and thus it may be a useful tool for analyzing immune responses.     

Reverse-Phase Protein Array: Technology,Application, Data Processing,and Integration

Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ∼240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.     

A laser ablation ICP‐MS based method for multiplexed immunoblot analysis: applications to manganese‐dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.)

Manganese (Mn) constitutes an essential co‐factor in the oxygen‐evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody‐based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) methods, based on data‐dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re‐supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn‐dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA‐ICP‐MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA‐ICP‐MS provides an analytical platform with high throughput appropriate for screening large collections of plants.     

Combination of microwell structures and direct oxygenation enables efficient and size‐regulated aggregate formation of an insulin‐secreting pancreatic β‐cell line

Spherical three‐dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell‐based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor‐intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen‐permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6‐m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (?32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0×105 cells/cm²) than that of normal confluent monolayers (1.0×105 cells/cm²). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS‐based culture system. Glucose‐responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell‐based PDMS culture system provides a promising method to form size‐regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:178–187, 2014     

Identification and characterization of Cryptococcus neoformans protein fractions that induce protective immune responses

Cryptococcus neoformans, the main causative agent of cryptococcosis, is a fungal pathogen that causes life‐threatening meningoencephalitis in immunocompromised patients. To date, there is no vaccine or immunotherapy approved to treat cryptococcosis. Cell‐ and antibody‐mediated immune responses collaborate to mediate optimal protection against C. neoformans infections. Accordingly, we identified cryptococcal protein fractions capable of stimulating cell‐ and antibody‐mediated immune responses and determined their efficacy to elicit protection against cryptococcosis. Proteins were extracted from C. neoformans and fractionated based on molecular mass. The fractions were then evaluated by immunoblot analysis for reactivity to serum extracted from protectively immunized mice and in cytokine recall assays for their efficacy to induce pro‐inflammatory and Th1‐type cytokine responses associated with protection. MS analysis revealed a number of proteins with roles in stress response, signal transduction, carbohydrate metabolism, amino acid synthesis, and protein synthesis. Immunization with select protein fractions containing immunodominant antigens induced significantly prolonged survival against experimental pulmonary cryptococcosis. Our studies support using the combination of immunological and proteomic approaches to identify proteins that elicit antigen‐specific antibody and Th1‐type cytokine responses. The immunodominant antigens that were discovered represent attractive candidates for the development of novel subunit vaccines for treatment and/or prevention of cryptococcosis.     

Suspension array technology: evolution of the flat-array paradigm.

Suspension arrays of microspheres analyzed using flow cytometry offer a new approach to multiplexed assays for large-scale screening applications. By optically encoding micron-sized polymer particles, suspension microarrays can be created to enable highly multiplexed analysis of complex samples. Each element in the array is comprised of a subpopulation of particles with distinct optical properties and each array element bears a different surface receptor. Nucleic acids, proteins, lipids or carbohydrates can serve as receptors to support the analysis of a wide range of biomolecular assemblies, and applications in genomic and proteomic research are being developed. Coupled with recent innovations for rapid serial analysis of samples, molecular analysis with microsphere arrays holds significant potential as a general analysis platform for both research and clinical applications.     

Unraveling mitotic protein networks by 3D multiplexed epitope drug screening

Three‐dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow comprises iterative antibody staining, high‐content 3D imaging, and machine learning for detection of mitoses. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, a basis for model‐based prediction of spatially resolved affinities of proteins. As a proof‐of‐concept, we mapped twelve epitopes in 3D‐cultured spheroids and investigated the network effects of twelve mitotic cancer drugs. Our approach reveals novel insights into spindle fragility and chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways. 3D SPECS's ability to map potential drug targets by multiplexed immunofluorescence in 3D cell culture combined with our automated high‐content assay will inspire future functional protein expression and drug assays.     

Comparison of results using the gel microdrop cytokine secretion assay with ELISPOT and intracellular cytokine staining assay

The gel microdrop (GMD) secretion assay involves encapsulating single cells in a biotinylated agarose matrix, addition of a streptavidin bridge, diffusion of a biotinylated capture antibody, and detection of secreted molecules using a fluorescently labeled reporter antibody. Using flow cytometry, encapsulated cells can be analyzed or recovered based on cell type and secretory profile. Using murine Th2 cell line D10.G4.1 as a model, we recently demonstrated the feasibility of using the GMD cytokine secretion assay combined with flow cytometry to detect IL-4-producing cells after stimulation with the mitogen, Con A. In addition, subpopulations of encapsulated cells secreting IL-4 were simultaneously characterized by immunophenotyping. We found good correlation between results using the GMD cytokine secretion assay and results with the standard ELISPOT and intracellular cytokine (ICC) assays. The GMD cytokine secretion assay permits simultaneous detection of secreted cytokine and determination of cell surface phenotype on viable, single cells. Moreover, using fluorescence activated cell sorting (FACS), secreting cells of interest can be sorted, recovered, and cultured for further studies.