Signaling Profiling Research Articles

Inactivation of Myostatin Delays Senescence via TREX1-SASP in Bovine Skeletal Muscle Cells.

The myostatin (MSTN) gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between MSTN and age-related diseases. It is unclear how MSTN and age-associated muscle loss in other animals are related. In this study, we utilized MSTN gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to MSTN and muscle cell senescence. The expression of MSTN was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, MSTN was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that MSTN influenced three prime repair exonuclease 1 (TREX1) expression through the SMAD2/3 complex. The downregulation of MSTN contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of MSTN in cell senescence in large animals.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6330.

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human iPSC-derived cardiomyocytes presented a renewed opportunity. Here, we conducted a comparative study, assessing cellular signaling through cardiac G protein-coupled receptors (GPCRs) in rat neonatal cardiomyocytes (RNCMs) and human induced pluripotent stem cell-derived cardiomyocytes. Genetically encoded biosensors were used to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) activities in both cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human cardiomyocytes. In line with this, bulk RNA-seq revealed key differences in the expression patterns of GPCRs, G proteins and downstream effector expression levels. Our study demonstrates that human stem cell-derived models of the cardiomyocyte offer distinct advantages for understanding cellular signaling in the heart.

Multicolor flow cytometry in clinical samples for platelet signaling assessment

BackgroundAvailability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. ObjectivesTo describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). MethodsBy comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI–related disease with granule secretion defect). ResultsThe range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. ConclusionIn complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.

Early response evaluation by single cell signaling profiling in acute myeloid leukemia

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phospho-ERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.

Dynamic Phosphotyrosine-Dependent Signaling Profiling in Living Cells by Two-Dimensional Proximity Proteomics.

Tyrosine phosphorylation (pTyr)-dependent signaling pathways play a vital role in various biological processes, which are spatiotemporally assembled and dynamically regulated on a minute scale by pTyr in living cells. Studying these pTyr-mediated signaling complexes is therefore challenging due to the highly dynamic nature of the protein complexes and the low abundance of pTyr. In this study, we adopted minute-resolution APEX2-based proximity labeling (PL) in living cells and Src SH2 superbinder-based pTyr peptide enrichment for simultaneously profiling these protein complexes and associated pTyr sites from the same affinity-purified sample. Upon different time courses of EGF stimulation of the living cells stably expressing APEX2-FLAG-GRB2, we constructed two-dimensional time-course curves for both interactome and tyrosine phosphoproteome. Well-annotated pTyr signaling complexes in EGFR signaling and located at the endosome were quantified with tightly correlated time-course curves for both interacting proteins and pTyr sites. Importantly, the correlated time-course curves for EGFR and endosomal HGS were well validated by targeted-parallel reaction monitoring (PRM)-MS analysis. Taking advantage of the high sensitivity of the PRM assay, the low-abundant pTyr peptide EGFR pY1110, which cannot be identified in the data-dependent acquisition (DDA) analysis, could be well quantified. Collectively, this two-dimensional proximity proteomic strategy is promising for comprehensively characterizing pTyr-mediated protein complexes with high sensitivity in living cells.

Use of Novel ebBRET Biosensors for Comprehensive Signaling Profiling of One Hundred Therapeutically Relevant Human GPCRs

Functional selectivity is the ability of a given GPCR to engage multiple signaling pathways, with distinct ligands of the given receptor displaying different efficacies in engaging receptor-coupled pathways. Full exploitation of functional selectivity in drug development will require an exhaustive description of the effectors that can be engaged by a given receptor, thus revealing receptor- and ligand-specific signaling signatures. Here, we describe the signaling profiles of 100 therapeutically relevant human GPCRs in response to their endogenous (or prototypical) ligands. Profiling was performed with 15 pathway-selective enhanced bystander bioluminescence resonance energy transfer (ebBRET) biosensors monitoring the activation of specific Gα proteins and βarrestins 1 and 2. The G protein biosensors represent a new generation of BRET-based sensors that measure the translocation of G protein effectors to the plasma membrane with no need for modifying the G proteins or the receptors. Over 1,500 dose‑response curves were generated, revealing a great diversity in GPCR coupling selectivity. Our data highlight that the Gi family displayed the highest general coupling while Gs and G12/13 families were less frequently engaged by the receptors tested. Certain GPCRs (17%) showed greater selectivity, with coupling restricted to a single G protein subtype or members of the same G protein family. Others showed broader activation profiles; specifically, 39%, 35% and 9% of GPCRs tested coupled to members of two, three and all four G protein families, respectively. Somewhat surprisingly, some receptors showed G protein subtype selectivity among the members of the same family. Of note, over 50 novel GPCR/G protein couplings were uncovered. In addition to highlighting receptor G protein coupling preferences, our data revealed that 78% of GPCRs recruited βarrestin 1 and/or 2. For certain GPCRs, co‑expression of GRK2 augmented (and even exposed) βarrestin engagement. Finally, in addition to the signaling profiling application, we demonstrated the versatility and usefulness of our ebBRET biosensor platform to study constitutive GPCR activity/inverse agonism, ligand- and SNP-induced biased signaling and cross-talk systems pharmacology. Overall, this work complements and enriches the current body of data on GPCR effector coupling. Moreover, it presents innovative tools allowing to further explore novel GPCR pharmacology. The resources provided in this study, combined to other signaling profiling and omic-scale datasets, will help deconvolute the complexities of GPCR biology and pharmacology and lead to innovative therapeutic exploitation of GPCRs.

Maternal high fat diet interacts with the maternal microbiome to expand IL-17 producing type 3 innate lymphoid cells in offspring.

Abstract Introduction: Previously, we demonstrated that maternal high fat diet (mHFD) exposure results in a unique pattern of microbial colonization in offspring and subsequent expansion of IL-17 producing type 3 innate lymphoid cells (ILC3). We hypothesized that in utero mechanisms involving the maternal microbiome were responsible for the phenotype in offspring. Methods: To assess if the maternal microbiome was responsible for altering the mHFD offspring phenotype, we delivered mHFD and regular diet (RD)-exposed pups at birth by C-section (CS) and exposed mHFD mothers to broad-spectrum antibiotics (ABX) throughout pregnancy. We cross-fostered the offspring to RD mothers at birth. Microbial colonization in 2-week-old offspring was determined by 16s-based rRNA analysis, intestinal cytokines were quantified by qRT-PCR and lamina propria cells (LP) were profiled by flow cytometry. To determine if innate immunity is altered prenatally by maternal diet, we delivered mHFD and RD offspring by CS and examined the small intestine (SI) at birth for cytokine expression, TLR signaling and LP profiling. Global MyD88 KO mice on mHFD or RD were also examined for the same endpoints. Results: mHFD offspring at 2-weeks showed an expansion of Firmicutes and IL-17 producing ILC3, independent of the mode of delivery. ABX in mHFD mothers ameliorated the mHFD offspring phenotype, with microbial colonization and ILC3 similar to controls. mHFD offspring had altered TLR signaling at birth in SI. ILC3 were expanded at birth in mHFD offspring (14.2% vs 9.5%). MyD88−/− offspring exposed to mHFD did not have an expansion of ILC3. Conclusion: mHFD prenatally alters the offspring microbiome and ILC3 and this effect is dependent on the maternal microbiome and TLR signaling.

Acute Effects of Cheddar Cheese Consumption on Circulating Amino Acids and Human Skeletal Muscle.

Cheddar cheese is a protein-dense whole food and high in leucine content. However, no information is known about the acute blood amino acid kinetics and protein anabolic effects in skeletal muscle in healthy adults. Therefore, we conducted a crossover study in which men and women (n = 24; ~27 years, ~23 kg/m2) consumed cheese (20 g protein) or an isonitrogenous amount of milk. Blood and skeletal muscle biopsies were taken before and during the post absorptive period following ingestion. We evaluated circulating essential and non-essential amino acids, insulin, and free fatty acids and examined skeletal muscle anabolism by mTORC1 cellular localization, intracellular signaling, and ribosomal profiling. We found that cheese ingestion had a slower yet more sustained branched-chain amino acid circulation appearance over the postprandial period peaking at ~120 min. Cheese also modestly stimulated mTORC1 signaling and increased membrane localization. Using ribosomal profiling we found that, though both milk and cheese stimulated a muscle anabolic program associated with mTORC1 signaling that was more evident with milk, mTORC1 signaling persisted with cheese while also inducing a lower insulinogenic response. We conclude that Cheddar cheese induced a sustained blood amino acid and moderate muscle mTORC1 response yet had a lower glycemic profile compared to milk.

High throughput pSTAT signaling profiling by fluorescent cell barcoding and computational analysis

Fluorescent cell barcoding (FCB) is a multiplexing technique for high-throughput flow cytometry (FCM). Although powerful in minimizing staining variability, it remains a subjective FCM technique because of inter-operator variability and differences in data analysis. FCB was implemented by combining two-dye barcoding (DyLight 350 plus Pacific Orange) with five-color surface marker antibody and intracellular staining for phosphoprotein signaling analysis. We proposed a robust method to measure intra- and inter-assay variability of FCB in T/B cells and monocytes by combining range and ratio of variability to standard statistical analyses. Data analysis was carried out by conventional and semi-automated workflows and built with R software. Results obtained from both analyses were compared to assess feasibility and reproducibility of FCB data analysis by machine-learning methods. Our results showed efficient FCB using DyLight 350 and Pacific Orange at concentrations of 0, 15 or 30, and 250 μg/mL, and a high reproducibility of FCB in combination with surface marker and intracellular antibodies. Inter-operator variability was minimized by adding an internal control bridged across matrices used as rejection criterion if significant differences were present between runs. Computational workflows showed comparable results to conventional gating strategies. FCB can be used to study phosphoprotein signaling in T/B cells and monocytes with high reproducibility across operators, and the addition of bridge internal controls can further minimize inter-operator variability. This FCB protocol, which has high throughput analysis and low intra- and inter-assay variability, can be a powerful tool for clinical trial studies. Moreover, FCB data can be reliably analyzed using computational software.

Abstract LB-004: Signaling signature profiling of cancer-associated RTK mutants

Abstract RTKs bind to a variety of signaling molecules, such as growth factors, hormones, cell surface and extracellular matrix proteins, and regulate many critical processes, including cell survival, proliferation, motility and differentiation. Importantly, dysregulated RTK activity can affect many of the processes above and lead to the development of numerous types of cancer. Consequently, RTKs are prime targets for new anti-cancer agents. Yet, our understanding of the complex biology of RTKs remains incomplete and, as a result, their full therapeutic potential is largely underexploited. Indeed, most methodologies currently used to screen for anti-RTK therapeutics revolve around wild type RTK kinase activity. Although useful, kinase activity-based assays overlook key determinants of ligand therapeutic efficacy, including kinetics, localization and pathway-biased signaling. In addition, numerous mutations conferring drug resistance have been described for different RTKs. Hence, RTK drug development strategies focused solely on wild type receptors risk delivering drugs displaying limited therapeutic efficacy and scope. We have developed a patented live-cell BRET-based biosensor platform that aims to address these current challenges and limitations via novel RTK mutant signaling profiling performed with real-time spatio-temporal capabilities. Specifically, the bioSensAll [TM] platform offers the following innovative features: Receptor localization: It has been described that intracellular EGFR is highly associated with disease progression, drug resistance and worse overall survival in numerous types of cancer. Targeting intracellular EGFR has therefore become an increasingly attractive anti-cancer strategy. This platform allows for the real-time spatio-temporal monitoring of the RTK internalization process and can eventually expose different trafficking features between mutant and wild type receptors. Broad spectrum ligand signaling profiling & HTS: The platform also offers a unique range of biosensors that permit for monitoring of ligand-induced signaling kinetics across various effector proteins/pathways and in intracellular compartments. Overall, this new spatio-temporal layer of data generated with untagged receptors allows us to confidently characterize and compare the effect of RTK mutations on ligand dynamics. Non-canonical G protein activity: A unique attribute of our mutant analysis consists of measuring the Galpha i/o subfamily activation induced by RTKs. This crosstalk between RTKs and classical GPCR effector proteins is a new paradigm and opens up a crucial pathway than can be exploited for anti-cancer drug development. The bioSensAll [TM] RTK biosensor platform offers a novel “multidimensional” angle to study RTK mutants found in various cancers. The ability to measure both spatial and temporal aspects of receptor trafficking and signaling along multiple pathways may expose novel anticancer strategies that are both tumor agnostic and mutation resistant. Citation Format: Guilhem Dugast, Stephan Schann, Florence Gross, Arturo Mancini. Signaling signature profiling of cancer-associated RTK mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-004.

Global Signaling Profiling in a Human Model of Tumorigenic Progression Indicates a Role for Alternative RNA Splicing in Cellular Reprogramming.

Intracellular signaling is controlled to a large extent by the phosphorylation status of proteins. To determine how human breast cells can be reprogrammed during tumorigenic progression, we profiled cell lines in the MCF10A lineage by phosphoproteomic analyses. A large cluster of proteins involved in RNA splicing were hypophosphorylated as cells progressed to a hyperplastic state, and then hyperphosphorylated after progression to a fully metastatic phenotype. A comprehensive transcriptomic approach was used to determine whether alterations in splicing factor phosphorylation status would be reflected in changes in mRNA splicing. Results indicated that the degree of mRNA splicing trended with the degree of tumorigenicity of the 4 cell lines tested. That is, highly metastatic cell cultures had the greatest number of genes with splice variants, and these genes had greater fluctuations in expression intensities. Genes with high splicing indices were mapped against gene ontology terms to determine whether they have known roles in cancer. This group showed highly significant associations for angiogenesis, cytokine-mediated signaling, cell migration, programmed cell death and epithelial cell differentiation. In summary, data from global profiling of a human model of breast cancer development suggest that therapeutics should be developed which target signaling pathways that regulate RNA splicing.

Abstract 3865: ActivSignal platform technology for direct multiplex analysis of signaling pathway activation profiles

Abstract The multiplex IPAD assay technology from ActivSignal monitors activity of 26 major signaling pathways by examining phosphorylation events or expression of more than 60 most relevant human and mouse protein targets. The technology uses antibodies against established and well known targets that reflect activities of canonical pathways. We utilize paired antibodies for each protein target for high specificity, and these pairs are carefully selected and tested. Only if both antibodies in a pair bind to their specific target molecule will the detection take place during the analysis phase - avoiding false hits. The IPAD assay can be used to profile samples made from cell culture, tissues, blood, etc. The IPAD assay requires only minute amounts of sample, which can be easily taken from your precious sample where available amounts may be limited. Applications include: Direct snapshot of signaling pathway activity rather than downstream effects. Understanding the physiological effects of a drug. What pathways are regulated in response to lead candidate drug? Does the drug of interest cause biologically significant secondary and off target effects? Evaluation of signaling pathway activities in response to shRNA, CRISPR, hormones, stresses and other treatments. Elucidating the signaling proteome in a medium-throughput format (96-well format). Profiling of tissue samples from patients and animals. Signaling profiling of tumor biopsies. In one application, our collaborators used the IPAD assay to understand signaling effects of a novel protein, TMIGD1, whose expression is markedly downregulated in renal cancer. The Activsignal platform analysis showed changes in activities of several major pathways, e.g. inhibition of cell cycle and cell proliferation after reintroduction of TMIGD1. In another application of our platform our collaborators studied the mechanism of action of a small molecule inhibitor that targets Hsp70. They used ActivSignal platform technology to profile signaling pathways in response to the inhibitor. They identified several pathways whose activities were affected by the inhibitor. ActivSignal is a breakthrough, low-cost tool for the analysis of signaling pathway activation profiles. It enables researchers to rapidly and efficiently gain insight into function of their favorite drug molecules, genes etc. Citation Format: Ilya Alexandrov, Irina Brandina, Nader Rahimi, Michael Sherman, Julia Yaglom, Rosana Meyer, Marwa Ali, Esma Ersoy. ActivSignal platform technology for direct multiplex analysis of signaling pathway activation profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3865.

High-Throughput Signaling Profiling in Blood Platelets by Multiplexed Phosphoflow Cytometry.

Multiplexed phosphoflow cytometry is a novel method that provides rapid and quantitative readouts on intracellular phosphoprotein signaling. In this approach, flow cytometry is combined with fluorescent cell barcoding (FCB) to facilitate high-throughput analyses of signaling events. After stimulation, fixed and permeabilized platelets are labeled with distinct dye intensities to create unique fluorescent signatures for individual samples. These uniquely labeled samples can be combined for simultaneous antibody staining and acquisition. During software analysis, multiplexed samples can be differentiated by their distinct fluorescence intensities and analyzed as if they had been acquired individually. Multiplexing eliminates intersample variation, increases statistical robustness, and allows 4-96 samples to be processed with no appreciable increase in antibody consumption or runtime. The method can be performed on washed platelets, platelet-rich plasma (PRP), and whole blood. Its inherent versatility can fulfil wide-ranging experimental requirements from simple dose titrations to complex pharmacologic screens.

Optimization and standardization of fluorescent cell barcoding for multiplexed flow cytometric phenotyping.

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4+ and CD8+ T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. Published 2017 by Wiley Periodicals, Inc., on behalf of International Society for Advancement of Cytometry. This article is a US government work and as such, is in the public domain in the United States of America.

Fluorescent Cell Barcoding As New Flow Cytometric Technique for Multiplexed Phenotyping and Signaling Profiling in Hematologic Patients

Fluorescent cell barcoding (FCB) is a high-throughput multiplexing technique, in which samples from one or more donors can be combined, minimizing staining variability, antibody consumption, and decreasing sample volumes needed. In this study, we optimized FCB technique for routine application in immunophenotyping, phosphoFlow, and intracellular cytokine detection. Human peripheral blood mononuclear cells (PBMCs) from healthy controls and patients were used for optimization, and CBD450 and CBD500, Pacific Orange succinimidyl ester, DyLight 350, and DyLight 800 were tested for barcoding 6 to 36 samples. Working concentrations ranging from 0 to 125 µg/mL were tested, and viability dye staining was also optimized to increase data robustness. We first measured fluorescence intensity (MFIs) of six serial dilutions (0, 0.75, 3.25, 12.5, 62.5 and 125 µg/mL) and the difference in intensity separating them. Second, a 3x3 matrix was prepared using three concentrations of two different dyes, and PMBCs were barcoded with nine different combinations of FCB dye concentrations. When gated with the dyes, nine lymphocyte populations were detected. A 4x(3x3) matrix was also designed using three different FCB dyes (CBD450, DyLight 800, and Pacific Orange) at various concentrations to barcode 36 samples simultaneously. Thus, each lymphocyte population was clearly identified. The separation of each population or purity of deconvolution is based on the distance between MFIs and CVs. When we calculated the purity of deconvolution in our experiments, human PBMCs displayed CVs of 10 - 25%. When MFIs were separated by 2-fold increase, barcoded populations could be identified with a good resolution, higher when MFIs were separated by 3- or more fold increase. The use of viability dyes as LIVE/DEAD Fixable Aqua viability dye together with the FCB showed increase of data robustness due to exclusion of dead cells from gating strategy. In combination with viability dye, we successfully performed a six-color phosphoFlow and a simple four-color stainings using the same FCB dye combinations and cytometer voltages, and also combining one healthy control and two different patient samples. Our methods using FCB dye alone and/or in combination with antibody staining should be useful to efficiently perform multiplex drug screening and lymphocyte characterization. FCB minimizes batches and technical variations, and also increases data robustness, thus improving the immune phenotyping of patients. DisclosuresYoung:GSK/Novartis: Research Funding.

B Cell Anergy Modulated by TLR1/2 and the miR-17∼92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4.

Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BCR Ig) display a particularly indolent disease course. Immunogenetic studies of the clonotypic BCR Ig of CLL subset #4 suggested a resemblance with B cells rendered anergic through chronic autoantigenic stimulation. In this article, we provide experimental evidence that subset #4 CLL cells show low IgG levels, constitutive ERK1/2 activation, and fail to either release intracellular Ca(2+) or activate MAPK signaling after BCR cross-linking, thus displaying a signature of B cell anergy at both biochemical and functional levels. Interestingly, TLR1/2 triggering restored BCR functionality, likely breaching the anergic state, and this was accompanied by induction of the miR-17∼92 cluster, whose members target critical BCR-associated molecules, including MAPKs. In conclusion, we demonstrate BCR anergy in CLL subset #4 and implicate TLR signaling and the miR-17∼92 cluster in the regulation of the anergic state. This detailed signaling profiling of subset #4 has implications for advanced understanding of the complex regulation of intracellular signaling pathways in CLL, currently a major therapeutic target of the disease.

Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324

Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML.BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer.In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment.Results:The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783).Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group.PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. [Display omitted] [Display omitted] [Display omitted] DisclosuresHellesøy:BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.

Modulation of sphingolipid metabolism with calorie restriction enhances insulin action in skeletal muscle

This study sought to investigate the effect of calorie restriction (CR) on skeletal muscle sphingolipid metabolism and its contribution to improved insulin action in rats after a 6-month feeding study. Twenty nine (29) male Fischer 344 rats were randomized to an ad libitum (AL) diet or 30% CR. Dietary intake, body weight and insulin sensitivity were monitored. After 6 months, skeletal muscle (vastus lateralis) was obtained for insulin signaling and lipid profiling. CR significantly decreased insulin and glucose levels and also altered the expression and activity of proteins involved in sphingolipid formation and metabolism. The quantities of ceramides significantly increased in CR animals (P<.05; n=14–15), while ceramide metabolism products (i.e., glycosphingolipids: hexosylceramides and lactosylceramides) significantly decreased (P<.05; n=14–15). Ceramide phosphates, sphingomyelins, sphingosine and sphingosine phosphate were not significantly different between AL and CR groups (P=ns; n=14–15). Lactosylceramide quantities correlated significantly with surrogate markers of insulin resistance (homeostasis model of assessment on insulin resistance) (r=0.7; P<.005). Products of ceramide metabolism (glycosphingolipids), known to interfere with insulin signaling at elevated levels, were significantly reduced in the skeletal muscle of CR animals. The increase in insulin sensitivity is associated with glycosphingolipid levels.

Multiplexed phosphospecific flow cytometry enables large‐scale signaling profiling and drug screening in blood platelets

Dissecting the signaling events that contribute to platelet activation will increase our understanding of platelet function and aid in the development of new antiplatelet agents. However, high-throughput methodology for the quantitative analysis of platelet signaling events is still lacking. To develop a high-throughput assay for the analysis of platelet signaling events in whole blood. We developed a fluorescent barcoding protocol to facilitate multiplexing and enable large-scale signaling profiling in platelets in whole blood. The methodology allowed simultaneous staining and acquisition of 24-96 samples in a single analysis tube with a standard flow cytometer. This approach significantly reduced experimental numbers, data acquisition time, and antibody consumption, while providing automated statistically rich quantitative data on signaling events. Using vasodilator-stimulated phosphoprotein (VASP), an established marker of platelet inhibition and antiplatelet drug therapy, we demonstrated that the assay could detect subtle changes in phosphoVASP-Ser157/239 in response to cAMP-elevating agents of varying potency and known modulators of the cAMP signaling cascade. The assay could be used with washed platelets or whole blood, analyzed immediately or frozen, without any significant change in assay performance. To demonstrate the usefulness of the assay as a drug discovery platform, we examined a prostaglandin screening library. Our screen of 70 prostaglandin derivatives revealed three previously uncharacterized lipids that stimulated phosphorylation of VASP-Ser157. Follow-up analyses demonstrated that these agents elevated intraplatelet cAMP and inhibited collagen-induced platelet aggregation. This novel method enables rapid, large-scale quantitative signaling profiling and compound screening in human platelets present in whole blood.