Multiplex Protein Assays Research Articles

Serum interleukin-6 and C-reactive protein are associated with survival in melanoma patients receiving immune checkpoint inhibition.

BackgroundInflammatory mediators, including acute phase reactants and cytokines, have been reported to be associated with clinical efficacy in patients with melanoma and other cancers receiving immune checkpoint inhibitors (ICI). Analyses of patient sera from three large phase II/III randomized ICI trials, one of which included a chemotherapy arm, were performed to assess whether baseline levels of C-reactive protein (CRP), interleukin-6 (IL-6) or neutrophil/lymphocyte (N/L) ratios were prognostic or predictive.Patients and methodsBaseline and on-treatment sera were analyzed by multiplex protein assays from immunotherapy-naïve patients with metastatic melanoma randomized 1:1 on the Checkmate-064 phase II trial of sequential administration of nivolumab followed by ipilimumab or the reverse sequence. Baseline sera, and peripheral blood mononuclear cells using automated cell counting, were analyzed from treatment-naïve patients who were BRAF wild-type and randomly allocated 1:1 to receive nivolumab or dacarbazine on the phase III Checkmate-066 trial, and from treatment-naïve patients allocated 1:1:1 to receive nivolumab, ipilimumab or both ipilimumab and nivolumab on the phase III Checkmate-067 trial.ResultsHigher baseline levels of IL-6 and the N/L ratio, and to a lesser degree, CRP were associated with shorter survival in patients receiving ICI or chemotherapy. Increased on-treatment levels of IL-6 in patients on the Checkmate-064 study were also associated with shorter survival. IL-6 levels from patients on Checkmate-064, Checkmate-066 and Checkmate-067 were highly correlated with levels of CRP and the N/L ratio.ConclusionIL-6, CRP and the N/L ratio are prognostic factors with higher levels associated with shorter overall survival in patients with metastatic melanoma receiving ICI or chemotherapy in large randomized trials. In a multi-variable analysis of the randomized phase III Checkmate-067 study, IL-6 was a significant prognostic factor for survival.

Serum-based multiplex protein assay for early detection of colorectal cancer and precancerous lesions in a FIT positive population.

e16145 Background: Colorectal cancer (CRC) is the second leading cancer worldwide in terms of incidence, 5-year prevalence and mortality for both women and men ages 45 years old and up. The current screening method for many countries with organized screening programs is the FIT test for fecal occult blood; however, this test can result in false positive rates as high as 65%. A FIT reflex test could reduce unnecessary colonoscopies while reducing wait times for those patients that need confirmatory colonoscopies the most. Methods: Danish FIT positive colonoscopy confirmed serum samples (n = 1,499) were divided into training and validation sets maintaining approximately equivalent percentages of clean colonoscopy (40%), low risk adenomas (16%), medium risk adenomas (19%), high risk adenomas (13%), stage I CRC (5%), stage II CRC (2%), stage III CRC (4%), and stage IV CRC (0.5%). Proteins were quantified by custom 16-plex immunoassays utilizing the Luminex xMAP platform. A support vector machine supervised machine learning algorithm was trained with the 16 biomarkers plus age and FIT concentration using 1,291 samples for the outcome medium risk adenoma, high risk adenoma, and CRC. Then this algorithm was tested on a blind 208 sample validation set. Results: The training set was 90% sensitive and 27% specific (AUC = 0.68) and the validation set was 93% sensitive and 21% specific (AUC = 0.63). The sensitivities of the validation by risk/stage was as follows: medium risk adenoma 91%, high risk adenomas 92%, stage I CRC 100%, stage II CRC 100%, stage III CRC 100%, stage IV CRC 93%. Conclusions: This study demonstrates feasibility of a novel blood-based multiplex protein immunoassay for use as a reflex to FIT positive results in population wide screening. It detected nearly all adenomas and carcinomas while reducing FIT false positives and thus unnecessary colonoscopies by more than 20%. A FIT reflex test could alleviate endoscopy burden experienced in countries with organized cancer screening programs, while providing better patient outcomes by detecting polyps and early-stage CRC with high sensitivity.

A thymic stromal lymphopoietin polymorphism may provide protection from asthma by altering gene expression

Genome-wide association studies have identified associations of the single nucleotide polymorphism rs1837253 in the thymic stromal lymphopoietin (TSLP) gene with asthma, allergic disease and eosinophilia. The TSLP gene encodes two isoforms, long and short, and previous studies have indicated functional differences between these two isoforms. We investigated the expression of these TSLP isoforms in response to a pro-inflammatory signal, and the role of the rs1837253 genotype in gene isoform regulation. We cultured nasal epithelial cells of asthmatic and non-asthmatic subjects and evaluated poly(I:C)-induced TSLP protein secretion using multiplex protein assays and gene expression profiles of the TSLP isoforms, and related genes using real-time qPCR. We correlated these profiles with rs1837253 genotype. Asthmatic nasal epithelial cells exhibited increased TSLP protein secretion compared with nasal epithelial cells from healthy controls. The long TSLP isoform was more responsive to poly(I:C) stimulation. Additionally, the minor T allele of rs1837253 was less inducible than the major C allele, suggesting differential regulation; this may explain the "protective" effects of the T allele in asthma. Our results provide important insights into the differential regulation and function of TSLP isoforms, including the role of TSLP rs1837253 polymorphisms in allergic inflammatory processes. The key finding on the influence of TSLP genetic variation on disease expression/endotype could provide basis for investigation into targeted biologics for anti-TSLP therapies.

Wound Fluid Cytokine Profile Following Bone Regeneration Procedures.

Clinical parameters available to evaluate early healing phases of bone regeneration procedures are limited. This study explores wound fluid (WF) content for molecular markers to differentiate wound healing responses in the early postoperative period after bone graft placement. Fifteen patients (50 ± 5 years old; 8 men) scheduled to receive tooth extraction and bone graft placement at maxillary nonmolar single-tooth sites were recruited. Primary wound closure was not intended at time of surgery. Gingival crevicular fluid from adjacent teeth or WF from surgical wound edges were collected (30 seconds) at baseline, at 3, 6, and 9 days, and at 1 and 4 months. Multiplex protein assay was used to determine concentration of various wound healing mediators. Immediately after surgery, 87% of surgical sites exhibited open wound. At day 9, mean wound exposure was 4.8 ± 0.4 mm. At 1 month, all wounds were clinically closed. The WF tripled in volume at day 3 and day 6 (P ≤ .05), compared with baseline gingival crevicular fluid, and gradually decreased as wounds closed. The WF concentrations of interleukin (IL)-6, placental growth factor, plasminogen activator inhibitor 1, insulin-like growth factor binding protein 1, and soluble cluster determinant 40 ligand were increased during early healing days, generally with peak concentration at day 6 (P ≤ .004). Conversely, WF concentrations of IL-18 and epidermal growth factor were decreased after surgery, generally not reaching baseline values until wound closure (P ≤ .008). In general, WF cytokine expression kinetics were concordant with wound closure dynamics (P ≤ .04). These results suggest that WF molecular markers such as IL-6, and to a lesser extent placental growth factor and IL-18, might help differentiate wound healing responses after bone regeneration procedures.

Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence.

Background: Blockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses. Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system. Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. Methods: We assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the sympathetic nervous system (SNS) was determinedby measuring noradrenaline, a major SNS mediator. Results: There were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra. Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls. Conclusion: These data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations and is therefore unlikely to further aggravate stroke-associated infection susceptibility through altered availability of these key anti-microbialmediators.

Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence

Background: Blockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses. Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system. Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. Methods: We assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the sympathetic nervous system (SNS) was determined by measuring noradrenaline, a major SNS mediator. Results: There were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra. Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls. Conclusion: These data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations and is therefore unlikely to further aggravate stroke-associated infection susceptibility through altered availability of these key anti-microbial mediators.

Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence.

Background: Blockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses. Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system. Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. Methods: We assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the sympathetic nervous system (SNS) was determinedby measuring noradrenaline, a major SNS mediator. Results: There were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra. Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls. Conclusion: These data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations and is therefore unlikely to further aggravate stroke-associated infection susceptibility through reduced availability of these key anti-microbialmediators.

Critical role of STAT5 tetramerization in the pathogenesis of experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis

Abstract Multiple sclerosis (MS) is an immune mediated disease that predominantly impacts the central nervous system (CNS). Current MS treatments have demonstrated the ability to reduce the rate of MS relapses; however, they are not effective at preventing disease progression. Elucidating the pathways that govern the pathogenesis of MS may identify molecular mediators, which can act as novel therapeutic targets to treat MS progression. STAT5 is a transcription factor critical for the generation, activation, and function of different immune cell types. Upon activation, STAT5 can form dimers or tetramers, which have distinct functions. Using a Stat5a-Stat5b double knock-in N-domain mutant mouse strain (DKI mice), in which STAT5 dimers are functional, but STAT5 tetramers cannot be formed, we demonstrated that DKI mice developed less severe experimental autoimmune encephalomyelitis (EAE), a model of MS, compared to WT littermates. Moreover, a portion of DKI mice did not develop EAE symptoms. Flow cytometric analysis of peripheral blood revealed that during the early phase of EAE, WT and DKI mice exhibited similar levels of immune cell expansion in the periphery. However, at the peak of EAE, DKI mice had fewer immune cells in the CNS. This suggests that STAT5 tetramers promote immune cell egress to the CNS or/and immune cell expansion within the CNS. In addition, multiplex protein assays established that the expression of a pro-inflammatory cytokine and several chemokines, including IL-1α, CCL3, CCL17, and CCL22, are reduced in DKI mice compared to WT mice, which may account for the reduced disease severity observed in DKI mice. Taken together, the data suggest that STAT5 tetramers plays a multifaceted role in promoting inflammation resulting in EAE.

RGal Increases Intestinal Epithelial Barrier Function in a PKC‐Dependent Manner and Drives an Inflammatory Gene Expression Profile

BackgroundLoss of intestinal epithelial barrier function has been linked to inflammatory bowel disease (IBD). We previously showed that the dietary fibre, rhamnogalacturonan (RGal), reduces inflammation in a murine model of DSS colitis. Additionally, apical treatment of Caco2 human intestinal epithelial cells with RGal increases transepithelial electrical resistance (TER) and accelerates wound‐healing, but the underlying mechanisms remain unknown.AimsWe aimed to determine the mechanisms of the RGal mediated increase in epithelial barrier function. (1) Given the ability of PKC to modulate barrier permeability we aimed to determine the role of PKC in the RGal‐mediated increase in epithelial barrier function. (2) We aimed to determine changes in gene expression following RGal treatment.Methods(1) To determine the role of PKC in the RGal‐induced increase in epithelial barrier function, Caco2 monolayers were mounted in Ussing Chambers, pre‐treated with pan protein kinase C (PKC), PKCz‐specific, or classical PKC inhibitors (GFX, PKCz pseudosubstrate or Go6976 respectively) and then treated with RGal. Change in TER and FITC‐dextran flux in response to RGal were assessed. (2) To determine the transcriptionally dependent effects of RGal on epithelial barrier function, Caco2 monolayers were treated apically for 6, 12 and 24h with RGal and RNAseq was performed. Genes with changes in expression more than 3‐fold were entered into Enrichr for pathway analysis. In order to confirm changes in gene expression from RNAseq, a multi‐array immunoassay was performed.Results(1) RGal (1 mg/mL) reduced FITC‐dextran flux by 52.0% (n=5, p<0.05) 30 min post‐treatment. The effect of RGal on macromolecular permeability was reduced with GFX pre‐treatment (500 nM) by 75.3% (n=5, p<0.01). However, the ability of GFX to block the RGal‐mediated increase in barrier function was not mimicked in monolayers pre‐treated with PKCz pseudosubstrate inhibitor (10 mM, n=6) or Go6976 (10 nM, n=5). (2) While RGal had no significant effect on tight junction gene expression 6h post‐treatment, it upregulated the expression of inflammatory response genes including neutrophil regulatory genes such as CXCL8. Consistent with gene expression data, multiplex protein assay revealed an upregulation in CXCL8 family chemokines and G‐CSF as early as 2h in cell lysates and 6h in the supernatant.ConclusionsThe RGal‐mediated increase in intestinal epithelial barrier function is dependent on PKC. Transcriptionally, RGal upregulates the expression of inflammatory mediators such as CXCL8. Understanding the mechanism of the RGal‐induced increase in intestinal epithelial barrier function may allow for leverage of these mechanisms to treat IBD.Support or Funding InformationProject supported by the NSERC (Canada), J.S. supported by an UGREF from the American Physiological Society.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Serum protein signature of coronary artery disease in type 2 diabetes mellitus

BackgroundCoronary artery disease (CAD) is the leading cause of morbidity and mortality in patients with type 2 diabetes mellitus (T2DM). The purpose of the present study was to discriminate the Indian CAD patients with or without T2DM by using multiple pathophysiological biomarkers.MethodsUsing sensitive multiplex protein assays, we assessed 46 protein markers including cytokines/chemokines, metabolic hormones, adipokines and apolipoproteins for evaluating different pathophysiological conditions of control, T2DM, CAD and T2DM with CAD patients (T2DM_CAD). Network analysis was performed to create protein-protein interaction networks by using significantly (p < 0.05) altered protein markers in each disease using STRING 10.5 database. We used two supervised analysis methods i.e., between class analysis (BCA) and principal component analysis (PCA) to reveals distinct biomarkers profiles. Further, random forest classification (RF) was used to classify the diseases by the panel of markers.ResultsOur two supervised analysis methods BCA and PCA revealed a distinct biomarker profiles and high degree of variability in the marker profiles for T2DM_CAD and CAD. Thereafter, the present study identified multiple potential biomarkers to differentiate T2DM, CAD, and T2DM_CAD patients based on their relative abundance in serum. RF classified T2DM based on the abundance patterns of nine markers i.e., IL-1β, GM-CSF, glucagon, PAI-I, rantes, IP-10, resistin, GIP and Apo-B; CAD by 14 markers i.e., resistin, PDGF-BB, PAI-1, lipocalin-2, leptin, IL-13, eotaxin, GM-CSF, Apo-E, ghrelin, adipsin, GIP, Apo-CII and IP-10; and T2DM _CAD by 12 markers i.e., insulin, resistin, PAI-1, adiponectin, lipocalin-2, GM-CSF, adipsin, leptin, Apo-AII, rantes, IL-6 and ghrelin with respect to the control subjects. Using network analysis, we have identified several cellular network proteins like PTPN1, AKT1, INSR, LEPR, IRS1, IRS2, IL1R2, IL6R, PCSK9 and MYD88, which are responsible for regulating inflammation, insulin resistance, and atherosclerosis.ConclusionWe have identified three distinct sets of serum markers for diabetes, CAD and diabetes associated with CAD in Indian patients using nonparametric-based machine learning approach. These multiple marker classifiers may be useful for monitoring progression from a healthy person to T2DM and T2DM to T2DM_CAD. However, these findings need to be further confirmed in the future studies with large number of samples.

Reproducibility and Crossplatform Validation of Reverse-Phase Protein Array Data.

Reverse-phase protein array (RPPA) technology is a high-throughput antibody- and microarray-based approach for the rapid profiling of levels of proteins and protein posttranslational modifications in biological specimens. The technology consumes small amounts of samples, can sensitively detect low-abundance proteins and posttranslational modifications, enables measurements of multiple signaling pathways in parallel, has the capacity to analyze large sample numbers, and offers robust interexperimental reproducibility. These features of RPPA experiments have motivated and enabled the use of RPPA technology in various biomedical, translational, and clinical applications, including the delineation of molecular mechanisms of disease, profiling of druggable signaling pathway activation, and search for new prognostic markers. Owing to the complexity of many of these applications, such as developing multiplex protein assays for diagnostic laboratories or integrating posttranslational modification-level data using large-scale proteogenomic approaches, robust and well-validated data are essential. There are many distinct components of an RPPA workflow, and numerous possible technical setups and analysis parameter options exist. The differences between RPPA platform setups around the world offer opportunities to assess and minimize interplatform variation. Crossplatform validation may also aid in the evaluation of robust, platform-independent protein markers of disease and response to therapy.

Risk assessment for indeterminate pulmonary nodules using a novel, plasma-protein based biomarker assay.

Background:The increase in lung cancer screening is intensifying the need for a noninvasive test to characterize the many indeterminate pulmonary nodules (IPN) discovered. Correctly identifying non-cancerous nodules is needed to reduce overdiagnosis and overtreatment. Alternatively, early identification of malignant nodules may represent a potentially curable form of lung cancer.Objective:To develop and validate a plasma-based multiplexed protein assay for classifying IPN by discriminating between those with a lung cancer diagnosis established pathologically and those found to be clinically and radiographically stable for at least one year.Methods:Using a novel technology, we developed assays for plasma proteins associated with lung cancer into a panel for characterizing the risk that an IPN found on chest imaging is malignant. The assay panel was evaluated with a cohort of 277 samples, all from current smokers with an IPN 4–30 mm. Subjects were divided into training and test sets to identify a Support Vector Machine (SVM) model for risk classification containing those proteins and clinical factors that added discriminatory information to the Veteran’s Affairs (VA) Clinical Factors Model. The algorithm was then evaluated in an independent validation cohort.Results:Among the 97 validation study subjects, 68 were grouped as having intermediate risk by the VA model of which the SVM model correctly identified 44 (65%) of these intermediate-risk samples as low (n=16) or high risk (n=28). The SVM model negative predictive value (NPV) was 94% and its sensitivity was 94%.Conclusion:The performance of the novel plasma protein biomarker assay supports its use as a noninvasive risk assessment aid for characterizing IPN. The high NPV of the SVM model suggests its application as a rule-out test to increase the confidence of providers to avoid aggressive interventions for their patients for whom the VA model result is an inconclusive, intermediate risk.

Impact of Human Adipose Tissue-Derived Stem Cells on Malignant Melanoma Cells in An In Vitro Co-culture Model.

This study focuses on the interactions of human adipose tissue-derived stem cells (ADSCs) and malignant melanoma cells (MMCs) with regard to future cell-based skin therapies. The aim was to identify potential oncological risks as ADSCs could unintentionally be sited within the proximity of the tumor microenvironment of MMCs. An indirect co-culture model was used to analyze interactions between ADSCs and four different established melanoma cell lines (G-361, SK-Mel-5, MeWo and A2058) as well as two low-passage primary melanoma cell cultures (M1 and M2). Doubling time, migration and invasion, angiogenesis, quantitative real-time PCR of 229 tumor-associated genes and multiplex protein assays of 20 chemokines and growth factors and eight matrix metalloproteinases (MMPs) were evaluated. Co-culture with ADSCs significantly increased migration capacity of G-361, SK-Mel-5, A2058, MeWo and M1 and invasion capacity of G-361, SK-Mel-5 and A2058 melanoma cells. Furthermore, conditioned media from all ADSC-MMC-co-cultures induced tube formation in an angiogenesis assay in vitro. Gene expression analysis of ADSCs and MMCs, especially of low-passage melanoma cell cultures, revealed an increased expression of various genes with tumor-promoting activities, such as CXCL12, PTGS2, IL-6, and HGF upon ADSC-MMC-co-culture. In this context, a significant increase (up to 5,145-fold) in the expression of numerous tumor-associated proteins could be observed, e.g. several pro-angiogenic factors, such as VEGF, IL-8, and CCL2, as well as different matrix metalloproteinases, especially MMP-2. In conclusion, the current report clearly demonstrates that a bi-directional crosstalk between ADSCs and melanoma cells can enhance different malignant properties of melanoma cells in vitro.

The impact of human adipose tissue-derived stem cells on breast cancer cells: implications for cell-assisted lipotransfers in breast reconstruction

BackgroundIn this study we evaluated the interactions of human adipose tissue-derived stem cells (ADSCs) and different human breast cancer cell lines (BRCAs) with regard to the safety of cell-assisted lipotransfers for breast reconstruction and a thereby unintended co-localization of ADSCs and BRCAs.MethodsADSCs were co-cultured with five different human BRCAs (MCF-7, MDA-MB-231, SK-BR-3, ZR-75-30, and EVSA-T) and primary BRCAs from one patient in a transwell system, and cell-cell-interactions were analyzed by assessing doubling time, migration and invasion, angiogenesis, quantitative real-time polymerase chain reaction (PCR) of more than 300 tumor-associated genes, and multiplex protein assays of 20 chemokines and growth factors and eight matrix metalloproteinases (MMPs). Results of co-culture were compared to those of the respective monoculture.ResultsQuantitative real-time PCR revealed remarkable changes in the expression of multiple tumor-associated genes in co-culture compared to monocultures of both ADSCs and BRCAs. Concomitantly, the concentration of several tumor-associated proteins, such as cytokines and MMPs, were strongly increased in co-culture. Furthermore, exclusively in co-culture with ADSCs, the different BRCAs were exposed to several important tumor-modulating proteins, such as CCL2, HGF, or interleukins.Co-culture did not significantly affect cellular proliferation of either ADSCs or BRCAs (p > 0.05). The migration of MCF-7 and MDA-MB-231 BRCAs was significantly increased in co-culture with ADSCs by a mean of 11% and 23%, respectively (p = 0.04 and 0.012), as well as that of ADSCs in co-culture with MDA-MB-231, ZR-75-30, and EVSA-T (+11–15%, p = 0.035–0.045). Co-culture with MDA-MB-231, SK-BR-3, and EVSA-T BRCAs significantly increased the invasive behavior of ADSCs by a mean of 24–41% (p = 0.014–0.039). There were no significant differences in the in vitro invasive properties of BRCAs in co-culture compared to monoculture. An in vitro angiogenesis assay revealed an increased tube formation of conditioned media from co-cultured BRCAs and ADSCs compared to the respective monocultures.ConclusionThis study further elucidates the possible interactions of primary human ADSCs with human BRCAs, pointing towards a potential increased oncological risk which should not be neglected when considering a clinical use of cell-assisted lipoaspirates in breast reconstruction.

Peripheral Immune Profile and Minimal Residual Disease (MRD) Burden Following Autologous Stem Cell Transplantation (ASCT) in Multiple Myeloma (MM)

BACKGROUND: The immune system plays an essential role in both promoting and inhibiting the growth of MM. Loss of anti-myeloma immunity involves altered activation and polarization of effector cells as well as immunosuppressive cytokines. Immune dysfunction associated with MM can be partially or totally reversed when patients achieve clinical remission. Detection of MRD has become an achievable goal in MM, and is a powerful predictor of progression-free survival and overall survival. We hypothesized that the immune profile of MRDpos patients is distinct from MRDneg patients.PATIENTS AND METHODS: We studied peripheral NK, NK-T and T cell distribution/activation, and measured bone marrow MRD status by flow cytometry in 30 newly diagnosed MM patients 60+ days after ASCT. Patients were divided in 2 groups based on κ/λ ratio and MM-plasma cell (MM-PC) distribution: MRDneg (n=6), with κ/λ ratio ≤1.8 and MM-PC≤15 per million or MRDpos (n=24), with MM-PC>15 per million. Plasma was also collected from 17 of these patients (5 MRDneg and 12 MRDpos) 60+ days after ASCT to quantify inflammatory cytokines, chemokines and growth factors by multiplex protein assay. Linear regression was used to determine association between each tested variable (25 by flow cytometry and 27 by multiplex protein assay) and MRD status. Unsupervised hierarchical cluster analysis was then applied to post-ASCT samples with selected variables that were differentially expressed between MRDneg and MRDpos patients (p<0.1) using complete linkage and Euclidean distances.RESULTS: More than a third of immune variables tested by flow cytometry (9/25) were significantly differential between MRDpos and MRDneg patients (p<0.1). Most significantly, MRDpos patients had fewer circulating CD56high NK cells than MRDneg (p=0.02). While NK cells had a propensity to be highly activated (90% NKG2D+) among MRDpos patients, they displayed an impaired killing phenotype with decreased KIR3DL1 expression compared with their MRDneg counterpart (p=0.01). NK-T and T cell distributions were not influenced by MRD status after ASCT, but phenotypically NK-T cells in MRDpos patients exhibited higher expression of NKG2D (p=0.05), but lacked NKG2A and Tim3 expression, indicative of greater mobilization. Plasma concentrations of all 27 soluble immune analytes tested were higher in MRDneg compared with MRDpos patients. Specifically, 2 pro-inflammatory cytokines [IL-12p70, IL-17] and 3 pro-angiogenic growth factors [FGF, PDGF, and VEGF] were highly correlated with MRD status (p<0.1). Unsupervised hierarchical clustering using all differentially expressed cellular and soluble immune markers showed better separation of MRDneg and MRDpos subgroups than using cellular or soluble immune markers separately (heat-map below).CONCLUSION: A comprehensive analysis of 52 immune variables assessing cytotoxic cell distribution, mobilization, killing potential, inflammatory status, immune polarization chemotaxis and angiogenesis revealed distinct differences in peripheral immunity between MRDpos and MRDneg MM patients after ASCT. Fewer circulating NK cells exhibiting a loss of killing potential characterized immune dysfunction among MRDpos patients. On the other hand, MRDneg patients more frequently displayed an inflammatory and pro-angiogenic cytokine profile. Taken together, these observations suggest that MRD represents a state of immune equilibrium where detectable or undetectable myeloma cells remain under surveillance by cellular and soluble immune mediators. As a next step, we are validating our observation in a larger prospective cohort by assessing peripheral immune profile as a predictor for MRD negativity. [Display omitted] DisclosuresSymanowski:Eli Lilly & Co: Consultancy; Ra Pharma: Consultancy; Caris Life Sciences: Consultancy; Endocyte: Consultancy. Avalos:Seattle Genetics: Membership on an entity’s Board of Directors or advisory committees. Usmani:BioPharma: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pharmacyclics: Research Funding; Onyx: Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Britsol-Myers Squibb: Consultancy, Research Funding; Millenium: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Array: Research Funding; Janssen: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Skyline: Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Takeda: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Bhutani:Takeda Oncology: Research Funding, Speakers Bureau; Prothena: Research Funding; Bristol-Myers Squibb: Speakers Bureau; Onyx, an Amgen subsidiary: Speakers Bureau.

NF-κb / STAT3 Signaling Pathway Collaboration in Diffuse Large B-Cell Lymphoma (DLBCL) Cell-of-Origin (COO) Subtypes: Resolution of Myeloid Subversion By Standard Induction Chemotherapy

Background. Germinal center B-cell (GCB) and activated B-cell (ABC) are two main cell-of-origin (COO) subtypes of diffuse large B-cell lymphomas(DLBCL). Patients presenting with the ABC-DLBCL subtype have inferior outcomes following standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy. Since ABC-DLBCL phenotype is characterized by marked activation of nuclear factor kappa B (NF-κB) signaling pathway, we hypothesized that inferior outcomes of patients with this COO subgroup treated with standard therapy were due to unresolved innate immune bias or myeloid subversion.Methods. Immunological profiles were established for 23 newly diagnosed DLBCL patients (n=13 ABC, n=10 GCB) prior to induction therapy (R-CHOP or R-EPOCH). Serum concentrations of 25 cytokines, chemokines and growth factors, were quantified by a multiplex protein assay. Peripheral distribution of myeloid cells (CD11b+ CD33+), myeloid maturation (HLA-DR+/-IL-4R+/-) and myeloid subversion [monocytic (m) and granulocytic (g) myeloid-derived suppressor cells (MDSCs)] were established by flow cytometry. These 31 immune variables were reassessed after completion of induction therapy for 16 DLBCL patients (n=10 ABC, n=6 GCB). Linear regression was used to determine the association between each tested variable and COO or modified international prognosis index (R-IPI). Unsupervised hierarchical cluster analysis was then applied to diagnosis samples with the selected variables that were differentially expressed between ABC- and GCB-DLBCL subtypes (p<0.1) using complete linkage and Euclidean distances. Paired t-test was used to compare each immune variable before and after chemotherapy.Results. At diagnosis, 14 of 25 immune analytes transcriptionally regulated by NF-κB were equally expressed between the two COO subgroups. ABC-DLBCL has lower PDGF-bb serum concentrations (p=.008). Two immune analytes regulated by the STAT3 signaling pathway, INFg-induced protein 10 (IP10) and IL-9 are differentially expressed among the two COO. While STAT3 positively regulates IP10, leading to elevated IP10 serum concentrations in ABC-DLBCL patients (p=0.003), it negatively regulates IL-9 leading to reduced IL-9 serum concentrations in this COO-subtype (p=0.05). Hierarchical clustering analysis of DLBCL patients at diagnosis shows that 9/13 ABC-DLBCL patients exhibit this STAT3 signature. STAT3 signaling pathway activation is also associated with myeloid immune subversion promoting gMDSC expansion in the ABC-DLBCL subtype (p=0.05). No correlations could be established between immunological bias at diagnosis in the 2 COO subtypes and R-IPI. Among the 16 patients who have completed chemotherapy (n=12 R-CHOP & n=4 R-EPOCH), none were refractory to induction therapy. ABC-DLBCL subtypes displayed the most profound immunological alterations compared with GCB-DLBCL, with decrease in serum concentrations of 7 immune analytes regulated by NF-κB (IL-6, IL-8, MIP1a, MIP1b, IL-1ra, GM-CSF and PDGF-bb). Standard chemotherapy leads to resolution of myeloid subversion and STAT3 bias as exemplified by normalization of the distribution of gMDSCs and IP10 and IL-9 serum concentrations between the two COO subgroups.Conclusion. Immune profiling of newly diagnosed DLBCL patients identified distinct biological signatures associated with COO subtypes. Highly enriched NF-κB signaling activation in ABC-DLBCL patients was associated with a STAT3-driven cytokine signature (NF-κB/STAT3 collaboration) and myeloid subversion. Standard induction chemotherapy normalized immunological bias across COO DLBCL subgroups. Longer follow up with clinical correlations are in progress to determine the significance of myeloid subversion and the NF-kB/STAT3 signature at diagnosis in DLBCL. DisclosuresAvalos:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Jacobs:Magellan Health: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau. Ghosh:Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; SGN: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genentech: Research Funding; TG Therapeutics: Research Funding.

Resistance exercise increases intramuscular NF-κb signaling in untrained males

The NF-κB signaling pathway regulates multiple cellular processes following exercise stress. This study aims to examine the effects of an acute lower-body resistance exercise protocol and subsequent recovery on intramuscular NF-κB signaling. Twenty-eight untrained males were assigned to either a control (CON; n=11) or exercise group (EX; n=17) and completed a lower-body resistance exercise protocol consisting of the back squat, leg press, and leg extension exercises. Skeletal muscle microbiopsies were obtained from the vastus lateralis pre-exercise (PRE), 1-hour (1H), 5-hours (5H), and 48-hours (48H) post-resistance exercise. Multiplex signaling assay kits (EMD Millipore, Billerica, MA, USA) were used to quantify the total protein (TNFR1, c-Myc) or phosphorylation status of proteins belonging to the NF-κB signaling pathway (IKKa/b, IkBα, NF-κB) using multiplex protein assay. Repeated measures ANOVA analysis was used to determine the effects of the exercise bout on intramuscular signaling at each time point. Additionally, change scores were analyzed by magnitude based inferences to determine a mechanistic interpretation. Repeated measures ANOVA indicated a trend for a two-way interaction between the EX and CON Group (p=0.064) for c-Myc post resistance exercise. Magnitude based inference analysis suggest a "Very Likely" increase in total c-Myc from PRE-5H and a "Likely" increase in IkBα phosphorylation from PRE-5H post-resistance exercise. Results indicated that c-Myc transcription factor is elevated following acute intense resistance exercise in untrained males. Future studies should examine the role that post-resistance exercise NF-κβ signaling plays in c-Myc induction, ribosome biogenesis and skeletal muscle regeneration.

Abstract 1853: Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib

Abstract Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibodies, which produce a detectable DNA amplicon only when they bind to the target protein. The resulting DNA amplicons are quantified by qPCR on the Biomark™ HD system. Data are analyzed using the Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format, which yields over 4,600 datapoints in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, we used it to profile the protein level changes in A431 cells treated with epidermal growth factor receptor (EGFR) inhibitor gefitinib. EGFR is a member of ErbB family of type I tyrosine kinases, whose overexpression and mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Using a multiplexed protein assay, we discovered some interesting co-expression patterns of proteins in single cells. We also demonstrated that TNFRSF10B was upregulated, while MET, GATA3, BRCA1, CCNB1, Ki-67 and CCNA2 were downregulated by gefitinib treatment. Notably, the reduction of the cell cycle-related proteins CCNB1 and CCNA2 and the cell proliferation marker Ki-67 was consistent with the inhibited cell growth by gefitinib. In addition, we measured mRNA expression to investigate the association between protein and mRNA levels in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis. Citation Format: Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. Single-cell multiplexed profiling of protein-level changes induced by EGFR inhibitor gefitinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1853.

Protein-based profiling of the immune response to uropathogenic Escherichia coli in adult patients immediately following hospital admission for acute cystitis.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are common infections in humans. Despite the substantial healthcare cost represented by these infections, the human immune response associated with the infection immediately following the onset of symptoms in patients remains largely undefined. We performed a prospective study aimed at defining the milieu of urinary cytokines in adult inpatients in the 24-48 h period immediately following hospital admission for acute cystitis due to UPEC. Urine samples, analyzed using 27-target multiplex protein assays, were used to generate immune profiles for patients and compared to age- and gender-matched healthy controls. The levels of multiple pro-inflammatory cytokines were significantly elevated in urine as a result of infection, an observation consistent with prior findings in murine models and clinical literature. We also identified significant responses for several novel factors not previously associated with the human response to UTI, including Interleukin (IL)-4, IL-7, IL-9, IL-17A, eotaxin, Granulocyte-macrophage colony-stimulating factor (GM-CSF) and several growth factors. These data establish crucial parallels between the human immune response to UPEC and murine model UTI studies, and emphasize the complex but poorly defined nature of the human immune response to UPEC, particularly in the immediate period following the onset of symptoms for acute cystitis.

Abstract B18: A simple method to prepare oligonucleotide-conjugated antibodies for multiplex protein detection

Abstract With rapid advances in nucleic acid detection technologies, genomics studies are being conducted in highly multiplexed formats. However, multiplex analysis is still a daunting task in the proteomics field; it would be of great interest to use genomics tools for protein detection. One way to accomplish this is to use oligonucleotide-conjugated antibodies to bind to specific protein targets, and to monitor conjugated-oligonucleotide levels rather than to measure protein levels directly. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method’s application in oligo extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and linked covalently with an azide-modified oligonucleotide. The reaction condition and purification process were optimized to achieve maximum yield and best performance in the functional test. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain a six-base complementary region at their 3′ prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. The template is then detected by qPCR on the Biomark HD system. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM and CASP3, had an estimated limit of detection (LOD) of about 0.06 pg/mL and a dynamic range of ≥5 logs. These antibody binders were used in multiplex protein detection in single cells using the C1™ system. The results in individual cells showed the expression of similar protein levels for CSTB and CASP3, but highly varied protein levels for EpCAM, CCNB1 and MUC1. This simple method for the preparation of oligonucleotide-conjugated antibodies is essential for the development of affordable multiplex protein assays, which has the potential to accelerate proteomics research. Citation Format: Haibiao Gong, Ilona Holcomb, Aik Ooi, Xiaohui Wang, Daniel Majonis, Marc Unger, Ramesh Ramakrishnan. A simple method to prepare oligonucleotide-conjugated antibodies for multiplex protein detection. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B18.