Single-cell Protein Analysis Research Articles

Innate immune response in COVID-19: single-cell multi-omics profile of NK lymphocytes in a clinical case series

BackgroundCOVID-19 pandemic caused by the Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) represents the biggest global health emergency in recent decades. The host immune response to SARS-CoV-2 seems to play a key role in disease pathogenesis and clinical manifestations, with Natural Killer (NK) lymphocytes being among the targets of virus-induced regulation.MethodsThis study performed a single-cell multi-omics analysis of transcripts and proteins of NK lymphocytes in COVID-19 patients, for the characterization of the innate immunological response to infection. NK cells were isolated from peripheral blood samples collected from adult subjects divided into 3 study groups: (1) non-infected subjects (Naïve group, n = 3), (2) post COVID-19 convalescent subjects (Healed group, n = 3) and (3) patients that were vaccinated against SARS-CoV-2 (Vaccine group, n = 3). Cells were then analysed by the BD Rhapsody System for the single-cell multi-omics investigation of transcriptome and membrane proteins.ResultsThe bioinformatic analysis identified 5 cell clusters which differentially expressed gene/protein markers, defining NK cell subsets as “Active NK cells” and “Mature NK cells”. Calculating the relative proportion of each cluster within patient groups, more than 40% of the Naïve group cell population was found to belong to Mature NKs, whereas more than 75% of the Vaccine group cell population belonged to the cluster of Active NKs. Regarding the Healed group, it seemed to show intermediate phenotype between Active and Mature NK cells. Differential expression of specific genes, proteins and signaling pathways was detected comparing the profile of the 3 experimental groups, revealing a more activated NK cell phenotype in vaccinated patients versus recovered individuals.ConclusionsThe present study detected differential expression of NK cell markers in relation to SARS-CoV-2 infection and vaccine administration, suggesting the possibility to identify key molecular targets for clinical-diagnostic use of the individual response to viral infection and/or re-infection.

Simple methodology to score micropattern quality and effectiveness.

Micropatterns (MPs) are widely used as a powerful tool to control cell morphology and phenotype. However, methods for determining the effectiveness of how well cells are controlled by the shape of MPs have been inconsistently used and studies rarely report on this topic, indicating lack of standardization. We introduce an evaluation score that quantitatively assesses the MP fabrication quality and effectiveness, which can be broadly used in conjunction with all currently available MP design types. This score uses four simple and quick steps: (i) scoring MP and (ii) background fabrication quality, (iii) defining the type(s) of MP of interest, and (iv) assigning so-called efficiency descriptors describing cell behavior. These steps are based on visual inspection and quick categorization of various aspects of MP fabrication quality and cell behaviour, presented in illustrations and microscopy image examples intended to serve as a reference "atlas". To illustrate the advantage of using this score, we determined differences in cell morphology and F-actin intensity between scored vs. non-scored cells. These measurements, which could be different in other studies, were chosen because both are understood as markers of cell phenotype and function. We combined intensity-calibrated immunofluorescence microscopy and image-based single cell protein analysis. Importantly, significant differences in cell morphology and cytoskeletal protein content between scored vs. non-scored cells were noted: the unconditional inclusion of all experimental read-outs (i.e., all MP data regardless of MP quality and effectiveness) into the final results significantly misjudged the experimental readouts vs. only including experimental read-outs of quality-controlled and effective MPs, identified by scoring. Specifically, non-scoring underestimated the F-actin intensity per cell and quantitative cellular morphometric descriptors circularity and solidity and overestimated aspect ratio. Scoring improved the precision of cellular readouts, advocating the use of a MP quality and efficiency score as a quantitative decision-supporting tool in deciding whether or not particular MPs should be used for experiments, saving time and money. This simple scoring methodology can be used for improving MP fabrication, comparing results across studies, benefiting basic science studies and potential future clinical use of MPs by introducing standardization.

Highly Multiplexing, Throughput and Efficient Single-Cell Protein Analysis with Digital Microfluidics.

Proteins as crucial components of cells are responsible for the majority of cellular processes. Sensitive and efficient protein detection enables a more accurate and comprehensive investigation of cellular phenotypes and life activities. Here, a protein sequencing method with high multiplexing, high throughput, high cell utilization, and integration based on digital microfluidics (DMF-Protein-seq) is proposed, which transforms protein information into DNA sequencing readout via DNA-tagged antibodies and labels single cells with unique cell barcodes. In a 184-electrode DMF-Protein-seq system, ≈1800 cells are simultaneously detected per experimental run. The digital microfluidics device harnessing low-adsorbed hydrophobic surface and contaminants-isolated reaction space supports high cell utilization (>90%) and high mapping reads (>90%) with the input cells ranging from 140 to 2000. This system leverages split&pool strategy on the DMF chip for the first time to overcome DMF platform restriction in cell analysis throughput and replace the traditionally tedious bench-top combinatorial barcoding. With the benefits of high efficiency and sensitivity in protein analysis, the system offers great potential for cell classification and drug monitoring based on protein expression at the single-cell level.

Abstract LB079: Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4

Abstract T cell-dependent anti-tumor activity unleashed by immunotherapies such as immune checkpoint therapy (ICT) can generate durable clinical responses. However, not all patients benefit, and epigenetic and transcriptional features required by T cells for effective ICT remain to be fully established. We previously documented that the transcription factor Bhlhe40 is upregulated by ICT in tumor specific CD4 and CD8 T cells in preclinical tumor models. We validated the relevance of this by ablating Bhlhe40 in both CD4 and CD8-T cells which rendered αPD-1 or αCTLA-4 ICT ineffective in tumor models normally responsive to ICT. Our findings extended beyond preclinical models as we recently found that human tumor-specific T cells recognizing mutant neoantigens display upregulation of Bhlhe40 and CRISPR-mediated Bhlhe40 deletion in human CD8 T cells recognizing the MART-1 tumor-antigen abrogates IFNG expression. Since we previously used CD4-Cre Bhlhe40f/f (B40ΔT) mice where Bhlhe40 was deleted in both CD4 and CD8 T cells, we could not definitively delineate how Bhlhe40 was regulating CD4 versus CD8 T cells under ICT. We thus used a model where Bhlhe40 is deleted specifically in CD8 T cells (B40ΔCD8). We found that like B40ΔT mice, B40ΔCD8 mice bearing the 1956 sarcoma were insensitive to αPD-1 ICT. While B40ΔT mice were also insensitive to αCTLA-4 ICT, interestingly, B40ΔCD8 mice displayed nearly normal tumor rejection upon αCTLA-4 ICT. Thus, Bhlhe40 is required in CD8 T cells for αPD-1 response, whereas is dispensable in CD8 T cells for αCTLA-4 efficacy. Single-cell protein and transcriptomic analysis of intratumoral immune-cells from ICT-treated control B40f/f, B40ΔCD8 and B40ΔT mice revealed overlapping and distinct changes to CD4 and CD8 T cells depending on which cells had Bhlhe40 deleted and which treatment was employed. With αPD-1 or αCTLA-4, defects in expression of IFN-γ, glycolytic enzyme transcripts and cytotoxic molecules, along with increased IL-10 production in CD4 and CD8 T cells from B40ΔT mice and in B40−/− CD8 T cells in B40ΔCD8 mice was observed. Whereas αCTLA-4-treated B40ΔT mice failed to reject tumors, addition of αIL-10R led to modest tumor growth inhibition, while with αPD-1 treatment, IL-10R blockade had little effect on tumor growth. We also noted that in control B40f/f mice, αPD-1 predominately altered CD8 T cells, whereas αCTLA-4 modified CD8 and CD4 T cells, including induction of an ICOS+ IFNγ+ CD4 T cell population expressing Bhlhe40. This population was largely preserved in B40ΔCD8 mice undergoing αCTLA-4 ICT. Analysis of intratumoral macrophages revealed profound defects in ICT-induced macrophage remodeling from CX3CR1+CD206+ to iNOS+ macrophages in B40ΔT mice lacking Bhlhe40 in CD4 and CD8 T cells. Although αCTLA-4-treated B40ΔCD8 mice reject their tumors, we observed a modestly higher frequency of CX3CR1+CD206+ macrophages and lower frequency of iNOS+ macrophages as compared to αCTLA-4-treated B40f/f mice. The highest percentage of CX3CR1+CD206+ macrophages and the fewest iNOS+ macrophages was observed in αPD-1-treated B40ΔCD8 mice that completely fail to reject tumors. These results reveal distinct requirements of Bhlhe40 in CD4 and CD8 T cells that depend on which ICT is used and further highlight non-redundant mechanisms of effective αPD-1 and αCTLA-4 Citation Format: Akata Saha, Alexander S. Shavkunov, Shailbala Singh, Avery J. Salmon, Sunita Keshari, Qi Miao, Brian T. Edelson, Cassian Yee, Ken Chen, Matthew Gubin. Cell-specific deletion of Bhlhe40 reveals distinct mechanisms underlying anti-PD-1 and anti-CTLA-4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB079.

High-Throughput Single-Cell, Single-Mitochondrial DNA Assay Using Hydrogel Droplet Microfluidics.

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis.

Photosensitive Hydrogel with Temperature-Controlled Reversible Nano-Apertures for Single-Cell Protein Analysis.

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

A nasal cell atlas reveals heterogeneity of tuft cells and their role in directing olfactory stem cell proliferation.

The olfactory neuroepithelium serves as a sensory organ for odors and forms part of the nasal mucosal barrier. Olfactory sensory neurons are surrounded and supported by epithelial cells. Among them, microvillous cells (MVCs) are strategically positioned at the apical surface, but their specific functions are enigmatic, and their relationship to the other specialized epithelial cells is unclear. Here, we establish that the family of MVCs comprises tuft cells and ionocytes in both mice and humans. Integrating analysis of the respiratory and olfactory epithelia, we define the distinct receptor expression of TRPM5+ tuft-MVCs compared with Gɑ-gustducinhigh respiratory tuft cells and characterize a previously undescribed population of glandular DCLK1+ tuft cells. To establish how allergen sensing by tuft-MVCs might direct olfactory mucosal responses, we used an integrated single-cell transcriptional and protein analysis. Inhalation of Alternaria induced mucosal epithelial effector molecules including Chil4 and a distinct pathway leading to proliferation of the quiescent olfactory horizontal basal stem cell (HBC) pool, both triggered in the absence of olfactory apoptosis. Alternaria- and ATP-elicited HBC proliferation was dependent on TRPM5+ tuft-MVCs, identifying these specialized epithelial cells as regulators of olfactory stem cell responses. Together, our data provide high-resolution characterization of nasal tuft cell heterogeneity and identify a function of TRPM5+ tuft-MVCs in directing the olfactory mucosal response to allergens.

Single-Cell Caspase-3 Measurement Using a Biomimetic Nanochannel.

Direct single-cell caspase-3 (Casp-3) analysis has remained challenging. A study of single-cell Casp-3 could contribute to revealing the fundamental pathogenic mechanisms in Casp-3-associated diseases. Here, a biomimetic nanochannel capable of single-cell sampling and ionic detection of intracellular Casp-3 is devised, which is established upon the installment of target-specific organic molecules (luc-DEVD) within the orifice of a glass nanopipette. The specific cleavage of luc-DEVD by Casp-3 could induce changes of inner-surface chemical groups and charge properties, thus altering the ionic response of the biomimetic nanochannel for direct Casp-3 detection. The practical applicability of this biomimetic nanochannel is confirmed by probing intracellular Casp-3 fluctuation upon drug stimulation and quantifying the Casp-3 evolution during induced apoptosis. This work realizes ionic single-cell Casp-3 analysis and provides a different perspective for single-cell protein analysis.

Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.

Single-cell analysis reveals a weak macrophage subpopulation response to Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) infection remains one of society's greatest human health challenges. Macrophages integrate multiple signals derived from ontogeny, infection, and the environment. This integration proceeds heterogeneously during infection. Some macrophages are infected, while others are not; therefore, bulk approaches mask the subpopulation dynamics. We establish a modular, targeted, single-cell protein analysis framework to study the immune response to Mtb. We demonstrate that during Mtb infection, only a small fraction of resting macrophages produce tumor necrosis factor (TNF) protein. We demonstrate that Mtb infection results in muted phosphorylation of p38 and JNK, regulators of inflammation, and leverage our single-cell methods to distinguish between pathogen-mediated interference in host signaling and weak activation of host pathways. We demonstrate that the inflammatory signal magnitude is decoupled from the ability to control Mtb growth. These data underscore the importance of developing pathogen-specific models of signaling and highlight barriers to activation of pathways that control inflammation.

Spatial Single Cell Analysis of Proteins in 2D Human Gastruloids Using Iterative Immunofluorescence.

During development, cell signaling instructs tissue patterning, the process by which initially identical cells give rise to spatially organized structures consisting of different cell types. How multiple signals combinatorially instruct fate in space and time remains poorly understood. Simultaneous measurement of signaling activity through multiple signaling pathways and of the cell fates they control is critical to addressing this problem. Here we describe an iterative immunofluorescence protocol and computational pipeline to interrogate pattern formation in a 2D model of human gastrulation with far greater multiplexing than is possible with standard immunofluorescence techniques. This protocol and computational pipeline together enable imaging followed by spatial and co-localization analysis of over 27 proteins in the same gastruloids. We demonstrate this by clustering single cell protein expression, using techniques familiar from scRNA-seq, and linking this to spatial position to calculate spatial distributions and cell signaling activity of different cell types. These methods are not limited to patterning in 2D gastruloids and can be easily extended to other contexts. In addition to the iterative immunofluorescence protocol and analysis pipeline, Support Protocols for 2D gastruloid differentiation and producing micropatterned multi-well slides are included. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Iterative immunofluorescence Basic Protocol 2: Computational analysis pipeline Support Protocol 1: Generating micropatterned multi-well slides Support Protocol 2: Differentiation of 2D gastruloids.

Development of constrictional microchannels and the recurrent neural network in single-cell protein analysis.

Introduction: As the golden approach of single-cell analysis, fluorescent flow cytometry can estimate single-cell proteins with high throughputs, which, however, cannot translate fluorescent intensities into protein numbers. Methods: This study reported a fluorescent flow cytometry based on constrictional microchannels for quantitative measurements of single-cell fluorescent levels and the recurrent neural network for data analysis of fluorescent profiles for high-accuracy cell-type classification. Results: As a demonstration, fluorescent profiles (e.g., FITC labeled β-actin antibody, PE labeled EpCAM antibody and PerCP labeled β-tubulin antibody) of individual A549 and CAL 27 cells were firstly measured and translated into protein numbers of 0.56 ± 0.43 × 104, 1.78 ± 1.06 × 106 and 8.11 ± 4.89 × 104 of A549 cells (ncell = 10232), and 3.47 ± 2.45 × 104, 2.65 ± 1.19 × 106 and 8.61 ± 5.25 × 104 of CAL 27 cells (ncell = 16376) based on the equivalent model of the constrictional microchannel. Then, the feedforward neural network was used to process these single-cell protein expressions, producing a classification accuracy of 92.0% for A549 vs. CAL 27 cells. In order to further increase the classification accuracies, as a key subtype of the recurrent neural network, the long short-term memory (LSTM) neural network was adopted to process fluorescent pulses sampled in constrictional microchannels directly, producing a classification accuracy of 95.5% for A549 vs. CAL 27 cells after optimization. Discussion: This fluorescent flow cytometry based on constrictional microchannels and recurrent neural network can function as an enabling tool of single-cell analysis and contribute to the development of quantitative cell biology.

Abstract 106: Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Heart Disease

Monocytes are innate immune cells that are defined by three subsets: classical (cMo), intermediate (iMo) and non-classical (nMo). How these subsets become more heterogeneous in phenotype and function in CVD progression remains elusive. We employed single-cell transcriptome and protein analysis to define how human monocytes differ in subjects with moderate to severe CVD. Peripheral blood mononuclear cells (PBMC) were obtained from 61 human subjects, undergoing coronary angiography as part of the Coronary Assessment of Virginia (CAVA) Cohort. Coronary atherosclerosis severity was quantified using Gensini Score (GS). We analyzed 487 immune-related genes and 49 surface proteins at the single cell level in PBMC from each subject using the BD-Rhapsody platform. WNN Seurat 4.0 was used for high dimensional analysis. Data was normalized using GLMM to control for covariates (age, sex, diabetes, and statin use). We identified cMo, iMo, nMo, plasmacytoid DC, and classical DC. We found a significant increase only in the frequencies of intermediate (iMo) monocytes in subjects with high CVD (GS>30) compared to subjects with low CVD (GS<6) ( p =0.004). Spearman correlation analysis with GS from each subject revealed a positive correlation with iMo frequencies (r=0.314, p =0.014) and further showed sex-dependent positive correlation in female patients (r=0.663, p =0.0037). Interestingly, cMo, thought to be potently atherogenic, did not correlate with CVD severity. Further analysis identified 3 iMo subsets, distinguished by expression of HLA-DR, CD86, CXCR3, and PD-L1. We found that frequencies of two of them, iMo_CXCR3 + PDL1 lo ( p =0.0003) and iMo_HLA-DR + CD86 int ( p =0.019), were associated with CVD severity. The iMo_CXCR3 + PDL1 lo immunoregulatory subset was positively correlated with GS in both females (r=0.66, p =0.004) and males (r=0.315, p =0.037). However, the iMo_HLA-DR + CD86 int subset was positively correlated only in females (r=0.55, p =0.022), illustrating substantial heterogeneity among monocyte subsets in a sex-dependent manner. In sum, we identified novel sex- and CVD-dependent iMo subsets in subjects with CVD. Future functional analysis of iMo populations should provide a deeper understanding of long-known sex-differences in CVD progression.

A single-cell rna sequencing analysis of alk1 and related bmp pathway proteins in osteoarthritis progression

A single-cell rna sequencing analysis of alk1 and related bmp pathway proteins in osteoarthritis progression

Prognostic value of integrated liquid biopsies in patients (pts) with metastatic urothelial cancer (mUC).

547 Background: Despite recent therapeutic advancements for patients with mUC, including checkpoint inhibitors (CPIs), anti-FGFR and antibody-drug conjugates (ADCs), biomarker data to predict therapeutic response and identify mechanisms of resistance remain limited. Ongoing randomized clinical trials evaluating the combination of CPIs and ADCs in these pts underscore the need to develop biomarkers to better understand who might best respond to these therapies. Here, we employ a liquid biopsy approach to molecularly characterize circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), including those isolated with Trop-2, the target of the novel drug Sacituzumab Govitecan and Datopotamab Deruxtecan. Methods: 104 blood samples serially collected from pts being treated for mUC were used for CTC collection using anti-EpCAM and Trop-2 antibodies in parallel. CTCs were isolated and stained for immune markers PD-L1 and HLA I using the VERSA (Versatile Exclusion-based Rare Sample Analysis) platform. Plasma was isolated for paired analysis of CTC and ctDNA content. CTCs were analyzed for enumeration and single-cell protein analysis. Overall survival (OS) was defined from time of sample collection, and survival analyses were performed using the Kaplan-Meier method. Results: We observed significantly shorter OS in pts with higher CTC burden (≥ 20 CTCs/7.5 mL blood) in samples captured with either EpCAM (median 34.1 vs 3.1 mo, P < 0.01) or Trop-2 (median 34.1 vs 6.2 months, P < 0.01). Furthermore, EpCAM and Trop-2 CTC burdens were higher at progression timepoints relative to responding or stable timepoints, when stratified according to treatment modality (table). Inter and intra-patient heterogeneous PD-L1 and HLA I expression and co-expression was observed on CTCs although no significant differences were seen globally between EpCAM and Trop-2 CTCs. In a cohort of pts treated with CPIs, we observed dynamic phenotypic changes in the ratio of HLA:PD-L1 that differed between EpCAM and Trop-2 CTCs. ctDNA analysis identified concordant increases in tumor fraction and acquired genomic mutations. When examined longitudinally across individual pts, these findings in tandem with changes in enumeration, suggest the emergence of different CTC subpopulations that may serve as biomarkers of resistance. Conclusions: We identify the prognostic significance of CTC enumeration with parallel EpCAM and Trop-2 CTC isolation and demonstrate the utility of a liquid biopsy assay that can detect pharmacodynamic changes in CTC burden and immune marker expression throughout the course of treatment. [Table: see text]

Strategies for Increasing the Depth and Throughput of Protein Analysis by plexDIA.

Accurate protein quantification is key to identifying protein markers, regulatory relationships between proteins, and pathophysiological mechanisms. Realizing this potential requires sensitive and deep protein analysis of a large number of samples. Toward this goal, proteomics throughput can be increased by parallelizing the analysis of both precursors and samples using multiplexed data independent acquisition (DIA) implemented by the plexDIA framework: https://plexDIA.slavovlab.net. Here we demonstrate the improved precisions of retention time estimates within plexDIA and how this enables more accurate protein quantification. plexDIA has demonstrated multiplicative gains in throughput, and these gains may be substantially amplified by improving the multiplexing reagents, data acquisition, and interpretation. We discuss future directions for advancing plexDIA, which include engineering optimized mass-tags for high-plexDIA, introducing isotopologous carriers, and developing algorithms that utilize the regular structures of plexDIA data to improve sensitivity, proteome coverage, and quantitative accuracy. These advances in plexDIA will increase the throughput of functional proteomic assays, including quantifying protein conformations, turnover dynamics, modifications states and activities. The sensitivity of these assays will extend to single-cell analysis, thus enabling functional single-cell protein analysis.

Notch-dependent cooperativity between myeloid lineages promotes Langerhans cell histiocytosis pathology.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Single-Cell Proteomics Preparation for Mass Spectrometry Analysis Using Freeze-Heat Lysis and an Isobaric Carrier.

Single-cell proteomics analysis requires sensitive, quantitatively accurate, widely accessible, and robust methods. To meet these requirements, the Single-Cell ProtEomics (SCoPE2) protocol was developed as a second-generation method for quantifying hundreds to thousands of proteins from limited samples, down to the level of a single cell. Experiments using this method have achieved quantifying over 3,000 proteins across 1,500 single mammalian cells (500-1,000 proteins per cell) in 10 days of mass spectrometer instrument time. SCoPE2 leverages a freeze-heat cycle for cell lysis, obviating the need for clean-up of single cells and consequently reducing sample losses, while expediting sample preparation and simplifying its automation. Additionally, the method uses an isobaric carrier, which aids protein identification and reduces sample losses. This video protocol provides detailed guidance to enable the adoption of automated single-cell protein analysis using only equipment and reagents that are widely accessible. We demonstrate critical steps in the procedure of preparing single cells for proteomic analysis, from harvesting up to injection to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Additionally, viewers are guided through the principles of experimental design with the isobaric carrier, quality control for both isobaric carrier and single-cell preparations, and representative results with a discussion of limitations of the approach.

Single-cell mass cytometry analysis reveals stem cell heterogeneity.

Cellular heterogeneity is fundamental to both developmental differentiation and disease establishment. Recent advances in high-throughput single-cell technology have been rapidly revolutionizing the resolution of our understanding of development and disease. However, while the study of single-cell transcriptomes is easily accessible, the analysis of single-cell proteomes is still in its infancy. In this study, we describe simultaneous profiling of multiple regulatory proteins at a single-cell level using mass cytometry or cytometry by time of flight. We develop mass cytometry reagents to study key transcription factors, signaling proteins and chromatin modifiers that regulate mouse embryonic stem cells. Our data reveal that the protein level of stem cell regulators significantly varies and that cell signaling pathways are extensively cross-activated across defined culture conditions of embryonic stem cells. In addition, the mass cytometry data enabled us to identify distinct multiple cell states of embryonic stem cells and determine their variation across culture conditions. We discuss the mass cytometry method, our results of the multi-protein analysis in embryonic stem cells and potential future perspectives for single-cell protein analysis.

Needle in a Haystack: A Pilot Study Combining Single-Cell Multiomics with Clinical NGS-MRD Sequencing to Search for Circulating Clonotypic Dedifferentiated Myeloma Cells

Multiple myeloma (MM) is a heterogenous disease in which clonal evolution or preexisting reservoirs of phenotypically divergent cells may be harbingers of therapeutic resistance. Prior research has suggested the presence of myeloma cells with immunophenotypic and expression profiles dedifferentiated from that of terminally differentiated plasma cells that may be found in circulation, marrow, or potentially other lymphoid compartments; however, this has not been conclusively demonstrated in patients. The identification of such clonotypic myeloma cells could lead to a better understanding of myeloma biology and resistance, and inform therapies targeted at these populations, and single-cell technologies provide the granularity to characterize them. Next-generation sequencing (NGS)-based measurable residual disease (MRD) clinical-grade assays (eg. clonoSEQ®, Adaptive Biosciences) identify individuals’ unique heavy and light chain sequences. Theoretically this sequencing data could be overlaid with research-grade single-cell multiomics to identify clusters of clonotypic cells with variable differentiation. In this pilot study using the Enhanced Single Cell Analysis with Protein Expression (ESCAPE) platform (Singleron Biotechnologies) we aimed to identify peripherally circulating clonotypic MM cells by their NGS-MRD sequence and simultaneously determine whether their multiomic profile reflected possible dedifferentiated clusters found in paired marrow. Using scRNA-Seq (10x Genomics) data previously generated at our site from bone marrow mononuclear cells we identified MM cell clusters enriched for differentially expressed genes known to be expressed in B-cells and plasmablasts. These genes included CD19, PAX5, BCL6, EZH2, STMN1, HMGB2, MKI67 and CD27. We next sought to assay the peripheral blood mononuclear cells (PBMC) of patients (n=2) with an overrepresentation of cells in these clusters. To evaluate both the transcriptomic and immunophenotypic profiles of the PBMCs at single-cell resolution we utilized Singleron's Immune Profiling panel on the ESCAPE platform. Using Singleron's MapSuite™ analysis pipeline, we annotated cell-type classifications, and highlighted cells within the mature B-cell/plasma cell differentiation spectrum (Figure A). Within this subset, we attempted to identify gene expression reflective of the previously identified "dedifferentiated” marrow clusters with specific genes of interest, but found that there was not significant overlap. Heatmaps comparing the 5 peripheral subclusters with marrow plasma cells demonstrate some concordance with the marrow subclusters, with examination of these overlaps ongoing, but these were not the "dedifferentiated” marrow clusters. The included patients also had clonoSEQ® identification sequences of heavy chain and/or light chain available. By cross-referencing the NGS-MRD sequence data (clonoSEQ®, Adaptive Biosciences), we were able to use ESCAPE-Seq to identify definitive clonotypic circulating MM cells within the PBMC samples at single cell granularity. Among PBMCs from sample 2 we were able to identify circulating clonotypic myeloma cells with 100% match to the IgH and IgK MRD sequences, which also represented the dominant V(D)J clonotype in peripheral circulating. Concomitant single-cell surface protein analysis of this clonotypic cell population demonstrated significant expression of CD38, without significant differential expression of CD138 nor surface markers of B-cells (Figure B). While these data are preliminary and studies are ongoing, we successfully leveraged clinical-level MRD sequencing data to inform single-cell multiomics, a potentially powerful translational research pathway for any hematologic malignancy in which NGS-MRD is clinically available. We definitively identified a small subset of circulating MM cells and used the ESCAPE platform to efficiently generate a multiomics profile. This profile suggested elevated CD38 expression without elevated CD138 expression, which previously has been associated with dedifferentiated plasma cells and plasmablasts, although further analyses are ongoing. In future studies, we will enrich for B-lymphoid spectrum cells prior to single-cell analytics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal