High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization.

Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.

Cell-type-specific signaling networks in heterocellular organoids.

Despite the widespread adoption of organoids as biomimetic tissue models, methods to comprehensively analyze cell-type-specific post-translational modification (PTM) signaling networks in organoids are absent. Here, we report multivariate single-cell analysis of such networks in organoids and organoid cocultures. Simultaneous analysis by mass cytometry of 28 PTMs in >1 million single cells derived from small intestinal organoids reveals cell-type- and cell-state-specific signaling networks in stem, Paneth, enteroendocrine, tuft and goblet cells, as well as enterocytes. Integrating single-cell PTM analysis with thiol-reactive organoid barcoding in situ (TOBis) enables high-throughput comparison of signaling networks between organoid cultures. Cell-type-specific PTM analysis of colorectal cancer organoid cocultures reveals that shApc, KrasG12D and Trp53R172H cell-autonomously mimic signaling states normally induced by stromal fibroblasts and macrophages. These results demonstrate how standard mass cytometry workflows can be modified to perform high-throughput multivariate cell-type-specific signaling analysis of healthy and cancerous organoids.

Uncovering axes of variation among single-cell cancer specimens.

While several tools have been developed to map axes of variation among individual cells, no analogous approaches exist for identifying axes of variation among multicellular biospecimens profiled at single-cell resolution. For this purpose, we developed 'phenotypic earth mover's distance' (PhEMD). PhEMD is a general method for embedding a 'manifold of manifolds', in which each datapoint in the higher-level manifold (of biospecimens) represents a collection of points that span a lower-level manifold (of cells). We apply PhEMD to a newly generated drug-screen dataset and demonstrate that PhEMD uncovers axes of cell subpopulational variation among a large set of perturbation conditions. Moreover, we show that PhEMD can be used to infer the phenotypes of biospecimens not directly profiled. Applied to clinical datasets, PhEMD generates a map of the patient-state space that highlights sources of patient-to-patient variation. PhEMD is scalable, compatible with leading batch-effect correction techniques and generalizable to multiple experimental designs.

The prospects of tumor chemosensitivity testing at the single-cell level.

Tumor chemosensitivity testing plays a pivotal role in the optimal selection of chemotherapeutic regimens for cancer patients in a personalized manner. High-throughput drug screening approaches have been developed but they failed to take into account intratumor heterogeneity and therefore only provided limited predictive power of therapeutic response to individual cancer patients. Single cancer cell drug sensitivity testing (SCC-DST) has been recently developed to evaluate the variable sensitivity of single cells to different anti-tumor drugs. In this review, we discuss how SCC-DST overcomes the obstacles of traditional drug screening methodologies. We outline critical procedures of SCC-DST responsible for single-cell generation and sorting, cell-drug encapsulation on a microfluidic chip and detection of cell-drug interactions. In SCC-DST, droplet-based microfluidics is emerging as an important platform that integrated various assays and analyses for drug susceptibility tests for individual patients. With the advancement of technology, both fluorescence imaging and label-free analysis have been used for detecting single cell-drug interactions. We also discuss the feasibility of integrating SCC-DST with single-cell RNA sequencing to unravel the mechanisms leading to drug resistance, and utilizing artificial intelligence to facilitate the analysis of various omics data in the evaluation of drug susceptibility. SCC-DST is setting the stage for better drug selection for individual cancer patients in the era of precision medicine.

Mass Cytometry Tags: Where Chemistry Meets Single-Cell Analysis.

Mass cytometry is a highly multiparametric proteomic technology that allows the measurement and quantification of nearly 50 markers with single-cell resolution. Mass cytometry reagents are probes tagged with metal isotopes of defined mass and act as reporters. Metals are detected using inductively coupled plasma time-of-flight mass spectrometry (ICP-TOF-MS). Many different types of mass-tag reagents have been developed to afford myriad applications. We have classified these compounds into polymer-based mass-tag reagents, nonpolymer-based mass-tag reagents, and inorganic nanoparticles. Metal-chelating polymers (MCPs) are widely used to profile and quantify cellular biomarkers; however, both the range of metals that can be detected and the metal signals have to be improved. Several strategies such as the inclusion of chelating agents or highly branched polymers may overcome these issues. Biocompatible materials such as polystyrene and inorganic nanoparticles are also of profound interest in mass cytometry. While polystyrene allows the inclusion of a wide variety of metals, the high metal content of inorganic nanoparticles offers an excellent opportunity to increase the signal from the metals to detect low-abundance biomarkers. Nonpolymer-based mass-tag reagents offer multiple applications: cell detection, cell cycle property determination, biomarker detection, and mass-tag cellular barcoding (MCB). Recent developments have been achieved in live cell barcoding by targeting proteins (CD45, b2m, and CD298), by using small and nonpolar probes or by ratiometric barcoding. From this perspective, the principal applications, strengths, and shortcomings of mass-tag reagents are highlighted, summarized, and discussed, with special emphasis on mass-tag reagents for MCB. Thereafter, the future perspectives of mass-tag reagents are discussed considering the current state-of-the-art technologies.

Alignment of single-cell trajectories to compare cellular expression dynamics.

Single-cell RNA sequencing and high-dimensional cytometry can be used to generate detailed trajectories of dynamic biological processes such as differentiation or development. Here we present cellAlign, a quantitative framework for comparing expression dynamics within and between single-cell trajectories. By applying cellAlign to mouse and human embryonic developmental trajectories, we systematically delineate differences in the temporal regulation of gene expression programs that would otherwise be masked.

Real-time fluorescence and deformability cytometry.

The throughput of cell mechanical characterization has recently approached that of conventional flow cytometers. However, this very sensitive, label-free approach still lacks the specificity of molecular markers. Here we developed an approach that combines real-time 1D-imaging fluorescence and deformability cytometry in one instrument (RT-FDC), thus opening many new research avenues. We demonstrated its utility by using subcellular fluorescence localization to identify mitotic cells and test for mechanical changes in those cells in an RNA interference screen.

Amperometry methods for monitoring vesicular quantal size and regulation of exocytosis release.

Chemical signaling strength during intercellular communication can be regulated by secretory cells through controlling the amount of signaling molecules that are released from a secretory vesicle during the exocytosis process. In addition, the chemical signal can also be influenced by the amount of neurotransmitters that is accumulated and stored inside the secretory vesicle compartment. Here, we present the development of analytical methodologies and cell model systems that have been applied in neuroscience research for gaining better insights into the biophysics and the molecular mechanisms, which are involved in the regulatory aspects of the exocytosis machinery affecting the output signal of chemical transmission at neuronal and neuroendocrine cells.

Removal of batch effects using distribution-matching residual networks.

MOTIVATION: Sources of variability in experimentally derived data include measurement error in addition to the physical phenomena of interest. This measurement error is a combination of systematic components, originating from the measuring instrument and random measurement errors. Several novel biological technologies, such as mass cytometry and single-cell RNA-seq (scRNA-seq), are plagued with systematic errors that may severely affect statistical analysis if the data are not properly calibrated. RESULTS: We propose a novel deep learning approach for removing systematic batch effects. Our method is based on a residual neural network, trained to minimize the Maximum Mean Discrepancy between the multivariate distributions of two replicates, measured in different batches. We apply our method to mass cytometry and scRNA-seq datasets, and demonstrate that it effectively attenuates batch effects. AVAILABILITY AND IMPLEMENTATION: our codes and data are publicly available at https://github.com/ushaham/BatchEffectRemoval.git. CONTACT: yuval.kluger@yale.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Automated cell type discovery and classification through knowledge transfer.

MOTIVATION: Recent advances in mass cytometry allow simultaneous measurements of up to 50 markers at single-cell resolution. However, the high dimensionality of mass cytometry data introduces computational challenges for automated data analysis and hinders translation of new biological understanding into clinical applications. Previous studies have applied machine learning to facilitate processing of mass cytometry data. However, manual inspection is still inevitable and becoming the barrier to reliable large-scale analysis. RESULTS: We present a new algorithm called utomated ell-type iscovery and lassification (ACDC) that fully automates the classification of canonical cell populations and highlights novel cell types in mass cytometry data. Evaluations on real-world data show ACDC provides accurate and reliable estimations compared to manual gating results. Additionally, ACDC automatically classifies previously ambiguous cell types to facilitate discovery. Our findings suggest that ACDC substantially improves both reliability and interpretability of results obtained from high-dimensional mass cytometry profiling data. AVAILABILITY AND IMPLEMENTATION: A Python package (Python 3) and analysis scripts for reproducing the results are availability on https://bitbucket.org/dudleylab/acdc . CONTACT: brian.kidd@mssm.edu or joel.dudley@mssm.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Joule Heating-Induced Dispersion in Open Microfluidic Electrophoretic Cytometry.

While protein electrophoresis conducted in capillaries and microchannels offers high-resolution separations, such formats can be cumbersome to parallelize for single-cell analysis. One approach for realizing large numbers of concurrent separations is open microfluidics (i.e., no microchannels). In an open microfluidic device adapted for single-cell electrophoresis, we perform 100s to 1000s of simultaneous separations of endogenous proteins. The microscope slide-sized device contains cells isolated in microwells located in a ∼40 µm polyacrylamide gel. The gel supports protein electrophoresis after concurrent in situ chemical lysis of each isolated cell. During electrophoresis, Joule (or resistive) heating degrades separation performance. Joule heating effects are expected to be acute in open microfluidic devices, where a single, high-conductivity buffer expedites the transition from cell lysis to protein electrophoresis. Here, we test three key assertions. First, Joule heating substantially impacts analytical sensitivity due to diffusive losses of protein out of the open microfluidic electrophoretic (EP) cytometry device. Second, increased analyte diffusivity due to autothermal runaway Joule heating is a dominant mechanism that reduces separation resolution in EP cytometry. Finally, buffer exchange reduces diffusive losses and band broadening, even when handling single-cell lysate protein concentrations in an open device. We develop numerical simulations of Joule heating-enhanced diffusion during electrophoresis and observe ∼50% protein loss out of the gel, which is reduced using the buffer exchange. Informed by analytical model predictions of separation resolution (with Joule heating), we empirically demonstrate nearly fully resolved separations of proteins with molecular mass differences of just 4 kDa or 12% (GAPDH, 36 kDa; PS6, 32 kDa) in each of 129 single cells. The attained separation performance with buffer exchange is relevant to detection of currently unmeasurable protein isoforms responsible for cancer progression.

Enhanced multiplexing in mass cytometry using osmium and ruthenium tetroxide species.

Mass cytometry facilitates high-dimensional, quantitative, single-cell analysis. The method for sample multiplexing in mass cytometry, called mass-tag cellular barcoding (MCB), relies on the covalent reaction of bifunctional metal chelators with intracellular proteins. Here, we describe the use of osmium and ruthenium tetroxides (OsO4 and RuO4 ) that bind covalently with fatty acids in the cellular membranes and aromatic amino acids in proteins. Both OsO4 and RuO4 rapidly reacted and allowed for MCB with live cells, crosslinked cells, and permeabilized cells. Given the covalent nature of the labeling reaction, isotope leaching was not observed. OsO4 and RuO4 were used in a 20-sample barcoding protocol together with palladium isotopes. As mass channels occupied by osmium and ruthenium are not used for antibody detection the number of masses effectively utilized in a single experiment is expanded. OsO4 and RuO4 can therefore be used as MCB reagents for a wide range of mass cytometry workflows. © 2016 International Society for Advancement of Cytometry.

A new tool for the quantitative analysis of dendritic filopodial motility.

Dendritic filopodia are tiny and highly motile protrusions formed along the dendrites of neurons. During the search for future presynaptic partners, their shape and size change dynamically, with a direct impact on the formation, stabilization and maintenance of synaptic connections both in vivo and in vitro. In order to reveal molecular players regulating synapse formation, quantitative analysis of dendritic filopodia motility is needed. Defining the length or the tips of these protrusions manually, however, is time consuming, limiting the extent of studies as well as their statistical power. Additionally, area detection based on defining a single intensity threshold can lead to significant errors throughout the image series, as these small structures often have low contrast in fluorescent images. To overcome these problems, the open access Dendritic Filopodia Motility Analyzer, a semi-automated ImageJ/Fiji plugin was created. Our method calculates the displacement of the centre of mass (CoM) within a selected region based on the weighted intensity values of structure forming pixels, selected by upper and lower intensity thresholds. Using synthetic and real biological samples, we prove that the displacement of the weighted CoM reliably characterizes the motility of dendritic protrusions. Additionally, guidelines to define optimal parameters of live cell recordings from dendritic protrusions are provided. © 2014 International Society for Advancement of Cytometry.

A deep profiler's guide to cytometry.

In recent years, advances in technology have provided us with tools to quantify the expression of multiple genes in individual cells. The ability to measure simultaneously multiple genes in the same cell is necessary to resolve the great diversity of cell subsets, as well as to define their function in the host. Fluorescence-based flow cytometry is the benchmark for this; with it, we can quantify 18 proteins per cell, at >10 000 cells/s. Mass cytometry is a new technology that promises to extend these capabilities significantly. Immunophenotyping by mass spectrometry provides the ability to measure >36 proteins at a rate of 1000 cells/s. We review these cytometric technologies, capable of high-content, high-throughput single-cell assays.

Plantaricin A, a cationic peptide produced by Lactobacillus plantarum, permeabilizes eukaryotic cell membranes by a mechanism dependent on negative surface charge linked to glycosylated membrane proteins.

Lactobacillus plantarum C11 releases plantaricin A (PlnA), a cationic peptide pheromone that has a membrane-permeabilizing, antimicrobial effect. We have previously shown that PlnA may also permeabilize eukaryotic cells, with a potency that differs between cell types. It is generally assumed that cationic antimicrobial peptides exert their effects through electrostatic attraction to negatively charged phospholipids in the membrane. The aim of the present study was to investigate if removal of the negative charge linked to glycosylated proteins at the cell surface reduces the permeabilizing potency of PlnA. The effects of PlnA were tested on clonal rat anterior pituitary cells (GH(4) cells) using patch clamp and microfluorometric techniques. In physiological extracellular solution, GH(4) cells are highly sensitive to PlnA, but the sensitivity was dramatically reduced in solutions that partly neutralize the negative surface charge of the cells, in agreement with the notion that electrostatic interactions are probably important for the PlnA effects. Trypsination of cells prior to PlnA exposure also rendered the cells less sensitive to the peptide, suggesting that negative charges linked to membrane proteins are involved in the permeabilizing action. Finally, pre-exposure of cells to a mixture of enzymes that split carbohydrate residues from the backbone of glycosylated proteins also impeded the PlnA-induced membrane permeabilization. We conclude that electrostatic attraction between PlnA and glycosylated membrane proteins is probably an essential first step before PlnA can interact with membrane phospholipids. Deviating glycosylation patterns may contribute to the variation in PlnA sensitivity of different cell types, including cancerous cells and their normal counterparts.

Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.

Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression.

Advancing cytometry for immunology.

Cytometry is a key technology for immunology. It allows researchers to scrutinize the cells of the immune system in molecular detail, and to assess phenotype and function at the level of individual cells, no matter how rare these cells may be. The International Society for the Advancement of Cytometry, ISAC, by way of its meetings, online resources and publications (e.g. Cytometry Part A and Current Protocols in Cytometry, which are all published by Wiley) track the ever advancing developments regarding cytometry instrumentation and reagents, and the analysis of complex data sets. In June this year in Leipzig, Germany, ISAC held its annual conference "CYTO 2012", a marketplace of innovation in cytometry.

[Value of percentage of highly fluorescent lymphocytic cells for rapidly assessing septicemia in tumor patients].

OBJECTIVE: To evaluate the value of percentage of highly fluorescent lymphocytic cells (HFLC%) for rapidly assessing septicemia in tumor patients. METHODS: Blood samples were collected from 130 patients with tumors (60 septicemia patients and 70 non-septicemia patients) and 80 healthy controls. HFLC% was analyzed with Sysmex XE-5000, the level of C-reactive protein (CRP) measured with a commercially available turbidimetric immunoassay kit and the level of procalcitonin (PCT) determined with a semiquantitative chromatographic immunoassay kit. The diagnostic values of HFLC% and CRP in septicemia were evaluated with ROC analysis. RESULTS: The values of HFLC% and CRP were significantly higher in the septicemia group than those in the non-septicemia and healthy groups (0.30% (0.10%-0.70%) vs 0.10% (0-0.20%), 0.10% (0-0.20%) ; 80.3 (28.5-129.5) vs 3.3 (1.4-41.4) , 1.4 (0.6-2.5) mg/L, all P < 0.01) . The ROC-AUCs for HFLC% and CRP for a diagnosis of septicemia were 0.72 (sensitivity 71.7%, specificity 58.7%) and 0.92 (sensitivity 96.7%, specificity 82.0%). Both of them could judge septicemia better. Additionally, HFLC% was correlated with the levels of PCT and CRP (r = 0.637, 0.241, both P < 0.01). CONCLUSIONS: HFLC% may be used as a rapid and simple auxiliary indicator in the diagnosis of septicemia in patients with tumors. And it is conducive to make an early diagnosis of septicemia and avoid unnecessary use of antibiotics.