Mass cytometry analysis of blood from peanut-sensitized tolerant and clinically allergic infants.

IgE-mediated food allergies in infants are a significant health concern, with peanut allergy being of particular interest due to its prevalence and severity. Among individuals who produce peanut-specific IgE some experience no adverse reaction on peanut consumption. This asymptomatic phenotype is known as sensitized tolerance. To elucidate the immune environment of peanut sensitized tolerant and clinically allergic one-year-olds, high-dimensional mass cytometry was conducted as part of the HealthNuts study. The resulting data includes peripheral blood mononuclear cells from 36 participants encompassing non-allergic, peanut sensitized with tolerance, and clinically peanut allergic infants. The raw mass cytometry data is described here and freely available for reuse through the Immunology Database and Analysis Portal (ImmPort). Additional allergy information and serum vitamin D levels of the participants were measured and are also included in the data upload. These high-dimensional mass cytometry data, when combined with clinical information, offer a broad immune profile of peanut allergic and sensitized tolerant infants.

Phenotypic Characterization by Single-Cell Mass Cytometry of Human Intrahepatic and Peripheral NK Cells in Patients with Hepatocellular Carcinoma.

Overall response rates of systemic therapies against advanced hepatocellular carcinoma (HCC) remain unsatisfactory. Thus, searching for new immunotherapy targets is indispensable. NK cells are crucial effectors and regulators in the tumor microenvironment and a determinant of responsiveness to checkpoint inhibitors. We revealed the landscape of NK cell phenotypes in HCC patients to find potential immunotherapy targets. Using single cell mass cytometry, we analyzed 32 surface markers on CD56dim and CD56bright NK cells, which included Sialic acid-binding immunoglobulin-type lectins (Siglecs). We compared peripheral NK cells between HCC patients and healthy volunteers. We also compared NK cells, in terms of their localizations, on an individual patient bases between peripheral and intrahepatic NK cells from cancerous and noncancerous liver tissues. In the HCC patient periphery, CD160+CD56dim NK cells that expressed Siglec-7, NKp46, and NKp30 were reduced, while CD49a+CD56dim NK cells that expressed Siglec-10 were increased. CD160 and CD49a on CD56dim NK cells were significantly correlated to other NK-related markers in HCC patients, which suggested that CD160 and CD49a were signature molecules. CD49a+ CX3CR1+ Siglec-10+ NK cells had accumulated in HCC tissues. Considering further functional analyses, CD160, CD49a, CX3CR1, and Siglec-10 on CD56dim NK cells may be targets for immunotherapies of HCC patients.

Cell fluorescence photoactivation as a method to select and study cellular subpopulations grown in mechanically heterogeneous environments.

A central challenge to the biology of development and disease is deciphering how individual cells process and respond to numerous biochemical and mechanical signals originating from the environment. Recent advances in genomic studies enabled the acquisition of information about population heterogeneity; however, these so far are poorly linked with the spatial heterogeneity of biochemical and mechanical cues. Whereas in vitro models offer superior control over spatiotemporal distribution of numerous mechanical parameters, researchers are limited by the lack of methods to select subpopulations of cells in order to understand how environmental heterogeneity directs the functional collective response. To circumvent these limitations, we present a method based on the use of photo convertible proteins, which when expressed within cells and activated with light, gives a stable fluorescence fingerprint enabling subsequent sorting and lysis for genomics analysis. Using this technique, we study the spatial distribution of genetic alterations on well-characterized local mechanical stimulation within the epithelial monolayer. Our method is an in vitro alternative to laser microdissection, which so far has found a broad application in ex vivo studies.

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.

A review of digital cytometry methods: estimating the relative abundance of cell types in a bulk of cells.

Due to the high cost of flow and mass cytometry, there has been a recent surge in the development of computational methods for estimating the relative distributions of cell types from the gene expression profile of a bulk of cells. Here, we review the five common 'digital cytometry' methods: deconvolution of RNA-Seq, cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT), CIBERSORTx, single sample gene set enrichment analysis and single-sample scoring of molecular phenotypes deconvolution method. The results show that CIBERSORTx B-mode, which uses batch correction to adjust the gene expression profile of the bulk of cells ('mixture data') to eliminate possible cross-platform variations between the mixture data and the gene expression data of single cells ('signature matrix'), outperforms other methods, especially when signature matrix and mixture data come from different platforms. However, in our tests, CIBERSORTx S-mode, which uses batch correction for adjusting the signature matrix instead of mixture data, did not perform better than the original CIBERSORT method, which does not use any batch correction method. This result suggests the need for further investigations into how to utilize batch correction in deconvolution methods.

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.

Alpha-Adrenergic Agonists Stimulate Fluid Secretion in Lacrimal Gland Ducts.

Purpose: The role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms. Methods: Fluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry. Results: Both norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [\(Ca_i^{2 + }\)]. Conclusions: Our results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of ß-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.

Highly functionalized ß-lactams and 2-ketopiperazines as TRPM8 antagonists with antiallodynic activity.

The cool sensor transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia, playing a key role in cold hypersensitivity associated to different peripheral neuropathies. Moreover, these channels are aberrantly expressed in different cancers, and seem to participate in tumor progression, survival and invasion. Accordingly, the search for potent and selective TRPM8 modulators attracted great interest in recent years. We describe new heterocyclic TRPM8 antagonist chemotypes derived from N-cloroalkyl phenylalaninol-Phe conjugates. The cyclization of these conjugates afforded highly substituted ß-lactams and/or 2-ketopiperazine (KP) derivatives, with regioselectivity depending on the N-chloroalkyl group and the configuration. These derivatives behave as TRPM8 antagonists in the Ca2+ microfluorometry assay, and confirmed electrophysiologically for the best enantiopure ß-lactams 24a and 29a (IC50, 1.4 and 0.8 µM). Two putative binding sites by the pore zone, different from those found for typical agonists and antagonists, were identified by in silico studies for both ß-lactams and KPs. ß-Lactams 24a and 29a display antitumor activity in different human tumor cell lines (micromolar potencies, A549, HT29, PSN1), but correlation with TRPM8 expression could not be established. Additionally, compound 24a significantly reduced cold allodynia in a mice model of oxaliplatin-induced peripheral neuropathy.

Intracellular pH regulation in mantle epithelial cells of the Pacific oyster, Crassostrea gigas.

Shell formation and repair occurs under the control of mantle epithelial cells in bivalve molluscs. However, limited information is available on the precise acid-base regulatory machinery present within these cells, which are fundamental to calcification. Here, we isolate mantle epithelial cells from the Pacific oyster, Crassostrea gigas and utilise live cell imaging in combination with the fluorescent dye, BCECF-AM to study intracellular pH (pHi) regulation. To elucidate the involvement of various ion transport mechanisms, modified seawater solutions (low sodium, low bicarbonate) and specific inhibitors for acid-base proteins were used. Diminished pH recovery in the absence of Na+ and under inhibition of sodium/hydrogen exchangers (NHEs) implicate the involvement of a sodium dependent cellular proton extrusion mechanism. In addition, pH recovery was reduced under inhibition of carbonic anhydrases. These data provide the foundation for a better understanding of acid-base regulation underlying the physiology of calcification in bivalves.

Wearing-off at the end of natalizumab dosing intervals is associated with low receptor occupancy.

OBJECTIVE: We aimed to investigate whether wearing-off symptoms at the end of the natalizumab dosing interval were associated with clinical and demographic patient characteristics or natalizumab receptor occupancy (RO) on leukocytes. METHODS: In this cross-sectional study of 40 patients with relapsing-remitting MS (RRMS) receiving natalizumab at the Department of Neurology, Haukeland University Hospital, we recorded clinical and demographic data including age, body mass index (BMI), working status, smoking habits, disease characteristics, treatment duration, vitamin D levels, and wearing-off symptoms. We quantified neurofilament light chain in serum and measured natalizumab RO in leukocyte subtypes by high-parameter mass cytometry. Associations with wearing-off symptoms were analyzed. RESULTS: Eight (20.0%) patients who reported regular occurrence of wearing-off symptoms, 9 (22.5%) who sometimes had wearing-off symptoms, and 23 (57.5%) who did not have wearing-off symptoms were evaluated. Patients who regularly had wearing-off symptoms had lower natalizumab RO than patients who reported having such symptoms sometimes or never. The former group also had higher BMI and higher frequency of sick leave. High BMI was associated with low RO. No other demographic or disease characteristics were associated with the phenomenon. CONCLUSIONS: Low RO may explain the wearing-off phenomenon observed in some patients with RRMS treated with natalizumab, and high BMI may be the underlying cause.

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.

Deformability-induced lift force in spiral microchannels for cell separation.

Cell sorting and isolation from a heterogeneous mixture is a crucial task in many aspects of cell biology, biotechnology and medicine. Recently, there has been an interest in methods allowing cell separation upon their intrinsic properties such as cell size and deformability, without the need for use of biochemical labels. Inertial focusing in spiral microchannels has been recognised as an attractive approach for high-throughput cell sorting for myriad point of care and clinical diagnostics. Particles of different sizes interact to a different degree with the fluid flow pattern generated within the spiral microchannel and that leads to particles ordering and separation based on size. However, the deformable nature of cells adds complexity to their ordering within the spiral channels. Herein, an additional force, deformability-induced lift force (FD), involved in the cell focusing mechanism within spiral microchannels has been identified, investigated and reported for the first time, using a cellular deformability model (where the deformability of cells is gradually altered using chemical treatments). Using this model, we demonstrated that spiral microchannels are capable of separating cells of the same size but different deformability properties, extending the capability of the previous method. We have developed a unique label-free approach for deformability-based purification through coupling the effect of FD with inertial focusing in spiral microchannels. This microfluidic-based purification strategy, free of the modifying immuno-labels, allowing cell processing at a large scale (millions of cells per min and mls of medium per minute), up to high purities and separation efficiency and without compromising cell quality.

Mapping lung cancer epithelial-mesenchymal transition states and trajectories with single-cell resolution.

Elucidating the spectrum of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) states in clinical samples promises insights on cancer progression and drug resistance. Using mass cytometry time-course analysis, we resolve lung cancer EMT states through TGFß-treatment and identify, through TGFß-withdrawal, a distinct MET state. We demonstrate significant differences between EMT and MET trajectories using a computational tool (TRACER) for reconstructing trajectories between cell states. In addition, we construct a lung cancer reference map of EMT and MET states referred to as the EMT-MET PHENOtypic STAte MaP (PHENOSTAMP). Using a neural net algorithm, we project clinical samples onto the EMT-MET PHENOSTAMP to characterize their phenotypic profile with single-cell resolution in terms of our in vitro EMT-MET analysis. In summary, we provide a framework to phenotypically characterize clinical samples in the context of in vitro EMT-MET findings which could help assess clinical relevance of EMT in cancer in future studies.

Design of an automated capillary electrophoresis platform for single-cell analysis.

Single-cell analysis of cellular contents by highly sensitive analytical instruments is known as chemical cytometry. A chemical cytometer typically samples one cell at a time, quantifies the cellular contents of interest, and then processes and reports that data. Automation adds the potential to perform this entire sequence of events with minimal intervention, increasing throughput and repeatability. In this chapter, we discuss the design considerations for an automated capillary electrophoresis-based instrument for assay of enzymatic activity within single cells. We describe the key requirements of the microscope base and capillary electrophoresis platforms. We also provide detailed protocols and schematic designs of our cell isolation, lysis, sampling, and detection strategies. Additionally, we describe our signal processing and instrument automation workflows. The described automated system has demonstrated single-cell throughput at rates above 100cells/h and analyte limits of detection as low as 10-20mol.

Quantification of fixed adherent cells using a strong enhancer of the fluorescence of DNA dyes.

Cell quantification is widely used in basic or applied research. The current sensitive methods of cell quantification are exclusively based on the analysis of non-fixed cells and do not allow the simultaneous detection of various cellular components. A fast, sensitive and cheap method of the quantification of fixed adherent cells is described here. It is based on the incubation of DAPI- or Hoechst 33342-stained cells in a solution containing SDS. The presence of SDS results in the quick de-staining of DNA and simultaneously, in an up-to-1,000-fold increase of the fluorescence intensity of the used dyes. This increase can be attributed to the micelle formation of SDS. The method is sufficiently sensitive to reveal around 50-70 human diploid cells. It is compatible with immunocytochemical detections, the detection of DNA replication and cell cycle analysis by image cytometry. The procedure was successfully tested for the analysis of cytotoxicity. The method is suitable for the quantification of cells exhibiting low metabolic activity including senescent cells. The developed procedure provides high linearity and the signal is high for at least 20 days at room temperature. Only around 90 to 120 minutes is required for the procedure's completion.

Cyclosis-mediated intercellular transmission of photosynthetic metabolites in Chara revealed with chlorophyll microfluorometry.

Symplastic interconnections of plant cells via perforations in adjoining cell walls (plasmodesmata) enable long-distance transport of photoassimilates and signaling substances required for growth and development. The pathways and features of intercellular movement of assimilates are often examined with fluorescent tracers whose molecular dimensions are similar to natural metabolites produced in photosynthesis. Chlorophyll fluorescence was recently found to be a sensitive noninvasive indicator of long-distance intracellular transport of physiologically produced photometabolites in characean internodes. The present work shows that the chlorophyll microfluorometry has a potential for studying the cell-to-cell transport of reducing substances released by local illumination of one internode and detected as the fluorescence increase in the neighbor internode. The method provides temporal resolution in the time frame of seconds and can be used to evaluate permeability of plasmodesmata to natural components released by illuminated chloroplasts. The results show that approximately one third of the amount of photometabolites released into the streaming cytoplasm during a 30-s pulse of local light permeates across the nodal complex with the characteristic time of ~ 10 s. The intercellular transport was highly sensitive to moderate elevations of osmolarity in the bath solution (150 mM sorbitol), which contrasts to the view that only transnodal gradients in osmolarity (and internal hydrostatic pressure) have an appreciable influence on plasmodesmal conductance. The inhibition of cell-to-cell transport was reversible and specific; the sorbitol addition had no influence on photosynthetic electron transport and the velocity of cytoplasmic streaming. The conductance of transcellular pores increased in the presence of the actin inhibitor cytochalasin D but the cell-to-cell transport was eventually suppressed due to the deceleration and cessation of cytoplasmic streaming. The results show that the permeability of plasmodesmata to low-molecular photometabolites is subject to upregulation and downregulation.

Recording SOCE Activity in Neurons by Patch-Clamp Electrophysiology and Microfluorometric Calcium Imaging.

Store-operated Ca2+ entry (SOCE) is a Ca2+ influx pathway at the plasma membrane that replenishes intracellular Ca2+ stores in response to depletion of Ca2+ stores. The SOC current, also known as the Ca2+ release-activated Ca2+ current (ICRAC), has a small conductance, which makes selective recording difficult. This challenge may be addressed using techniques based on identification of Ca2+ influx patch-clamp electrophysiological recording and measurement of cytoplasmic Ca2+ accumulation with Ca2+-sensitive fluorophores. Here, we describe specific methods for studying SOCE using these approaches in rat dorsal root ganglion neurons.