Fluorescence-activated Cell Sorting Assay Research Articles

In-depth organic mass cytometry reveals differential contents of 3-hydroxybutanoic acid at the single-cell level

Comprehensive single-cell metabolic profiling is critical for revealing phenotypic heterogeneity and elucidating the molecular mechanisms underlying biological processes. However, single-cell metabolomics remains challenging because of the limited metabolite coverage and inability to discriminate isomers. Herein, we establish a single-cell metabolomics platform for in-depth organic mass cytometry. Extended single-cell analysis time guarantees sufficient MS/MS acquisition for metabolite identification and the isomers discrimination while online sampling ensures the high-throughput of the method. The largest number of identified metabolites (approximately 600) are achieved in single cells and fine subtyping of MCF-7 cells is first demonstrated by an investigation on the differential levels of 3-hydroxybutanoic acid among clusters. Single-cell transcriptome analysis reveals differences in the expression of 3-hydroxybutanoic acid downstream antioxidative stress genes, such as metallothionein 2 (MT2A), while a fluorescence-activated cell sorting assay confirms the positive relationship between 3-hydroxybutanoic acid and target proteins; these results suggest that the heterogeneity of 3-hydroxybutanoic acid provides cancer cells with different ability to resist surrounding oxidative stress. Our method paves the way for deep single-cell metabolome profiling and investigations on the physiological and pathological processes that occur during cancer.

Atranorin inhibits Zika virus infection in human glioblastoma cell line SNB-19 via targeting Zika virus envelope protein

BackgroundZika virus (ZIKV) is a single-stranded RNA flavivirus transmitted by mosquitoes. Its infection is associated with neurological complications such as neonatal microcephaly and adult Guillain–Barré syndrome, posing a serious threat to the health of people worldwide. Therefore, there is an urgent need to develop effective anti-ZIKV drugs. Atranorin is a lichen secondary metabolite with a wide range of biological activities, including anti-inflammatory, antibacterial and antioxidant, etc. However, the antiviral activity of atranorin and underlying mechanism has not been fully elucidated. PurposeWe aimed to determine the anti-ZIKV activity of atranorin in human glioma cell line SNB-19 and investigate the potential mechanism from the perspective of viral life cycle and the host cell functions. MethodsWe first established ZIKV-infected human glioma cells (SNB-19) model and used Western Blot, RT-qPCR, immunofluorescence, fluorescence-activated cell sorting (FACS) and plaque assay to evaluate the anti-ZIKV activity of atranorin. Then we assessed the regulation effect of atranorin on ZIKV induced IFN signal pathway activation by RT-qPCR. Afterward, we introduced time-of-addition assay, viral adsorption assay, viral internalization assay and transferrin uptake assay to define which step of ZIKV lifecycle is influenced by atranorin. Finally, we performed virus infectivity assay, molecular docking and thermal shift assay to uncover the target protein of atranorin on ZIKV. ResultsOur study showed that atranorin could protect SNB-19 cells from ZIKV infection, as evidenced by inhibited viral protein expression and progeny virus yield. Meanwhile, atranorin attenuated the activation of IFN signal pathway and downstream inflammatory response that induced by ZIKV infection. The results of time-of-addition assay indicated that atranorin acted primarily by disturbing the viral entry process. After ruling out the effect of atranorin on AXL receptor tyrosine kinase (AXL) dependent virus adsorption and clathrin-mediated endocytosis, we confirmed that atranorin directly targeted the viral envelope protein and lowered ZIKV infectivity by thermal shift assay and virus infectivity assay respectively. ConclusionWe found atranorin inhibits ZIKV infection in SNB-19 cells via targeting ZIKV envelope protein. Our study provided an experimental basis for the further development of atranorin and a reference for antiviral drug discovery from natural resources.

Cysteine and glycine-rich protein 2 retards platelet-derived growth factor-BB-evoked phenotypic transition of airway smooth muscle cells by decreasing YAP/TAZ activity.

Cysteine and glycine-rich protein 2 (Csrp2) has emerged as a key factor in controlling the phenotypic modulation of smooth muscle cells. The phenotypic transition of airway smooth muscle cells (ASMCs) is a pivotal step in developing airway remodeling during the onset of asthma. However, whether Csrp2 mediates the phenotypic transition of ASMCs in airway remodeling during asthma onset is undetermined. This work aimed to address the link between Csrp2 and the phenotypic transition of ASMCs evoked by platelet-derived growth factor (PDGF)-BB in vitro. The overexpression or silencing of Csrp2 in ASMCs was achieved through adenovirus-mediated gene transfer. The expression of mRNA was measured by quantitative real-time-PCR. Protein levels were determined through Western blot analysis. Cell proliferation was detected by EdU assay and Calcein AM assays. Cell cycle distribution was assessed via fluorescence-activated cell sorting assay. Cell migration was evaluated using the scratch-wound assay. The transcriptional activity of Yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) was measured using the luciferase reporter assay. A decline in Csrp2 level occurred in PDGF-BB-stimulated ASMCs. Increasing Csrp2 expression repressed the PDGF-BB-evoked proliferation and migration of ASMCs. Moreover, increasing Csrp2 expression impeded the phenotypic change of PDGF-BB-stimulated ASMCs from a contractile phenotype into a synthetic/proliferative phenotype. On the contrary, the opposite effects were observed in Csrp2-silenced ASMCs. The activity of YAP/TAZ was elevated in PDGF-BB-stimulated ASMCs, which was weakened by Csrp2 overexpression or enhanced by Csrp2 silencing. The YAP/TAZ activator could reverse Csrp2-overexpression-mediated suppression of the PDGF-BB-evoked phenotypic switching of ASMCs, while the YAP/TAZ suppressor could dimmish Csrp2-silencing-mediated enhancement on PDGF-BB-evoked phenotypic switching of ASMCs. In summary, Csrp2 serves as a determinant for the phenotypic switching of ASMCs. Increasing Csrp2 is able to impede PDGF-BB-evoked phenotypic change of ASMCs from a synthetic phenotype into a synthetic/proliferative phenotype through the effects on YAP/TAZ. This work implies that Csrp2 may be a key player in airway remodeling during the onset of asthma.

Activation of coagulation FXI promotes endothelial inflammation and amplifies platelet activation in a nonhuman primate model of hyperlipidemia

BackgroundHyperlipidemia is associated with chronic inflammation and thromboinflammation. This is an underlying cause of several cardiovascular diseases, including atherosclerosis. In diseased blood vessels, rampant thrombin generation results in the initiation of the coagulation cascade, activation of platelets, and endothelial cell dysfunction. Coagulation factor (F) XI represents a promising therapeutic target to reduce thromboinflammation, as it is uniquely positioned at an intersection between inflammation and thrombin generation. ObjectivesThis study aimed to investigate the role of FXI in promoting platelet and endothelial cell activation in a model of hyperlipidemia. MethodsNonhuman primates (NHPs) were fed a standard chow diet (lean, n = 6) or a high-fat diet (obese, n = 8) to establish a model of hyperlipidemia. Obese NHPs were intravenously administered a FXI blocking antibody (2 mg/kg) and studied at baseline and at 1, 7, 14, 21, and 28 days after drug administration. Platelet activation and inflammatory markers were measured using fluorescence-activated cell sorting or enzyme-linked immunosorbent assay. Molecular imaging was used to quantify vascular cell adhesion molecule 1 (VCAM-1) expression at the carotid bifurcation. ResultsObese NHPs demonstrated increased sensitivity for platelet P-selectin expression and phosphatidylserine exposure in response to platelet GPVI or PAR agonists compared with lean NHPs. Obese NHPs exhibited elevated levels of C-reactive protein, cathepsin D, and myeloperoxidase compared with lean NHPs. Following pharmacological inhibition of FIX activation by FXIa, platelet priming for activation by GPVI or PAR agonists, C-reactive protein levels, and endothelial VCAM-1 levels were reduced in obese NHPs. ConclusionFXI activation promotes the proinflammatory phenotype of hyperlipidemia by priming platelet activation and inciting endothelial cell dysfunction.

Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing

Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest.

Myelin oligodendrocyte glycoprotein antibody-associated disease as a novel presentation of central nervous system autoimmunity in a pediatric patient with Wiskott-Aldrich syndrome

BackgroundWiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene that leads to increased susceptibility to infections, thrombocytopenia, eczema, malignancies, and autoimmunity. Central nervous system (CNS) autoimmune manifestations are uncommon.Case PresentationWe describe the case of a five-year-old boy with refractory thrombocytopenia and iron deficiency anemia who developed relapsing bilateral optic neuritis. Myelin oligodendrocyte glycoprotein antibody (MOG-IgG) via serum fluorescence-activated cell sorting assay was positive (titer 1:100), confirming a diagnosis of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). At age six, molecular panel testing for genes associated with primary immunodeficiency identified a missense WAS gene variant. He was subsequently found to have decreased WAS protein expression, consistent with a diagnosis of WAS.ConclusionsThis case expands the reported spectrum of CNS autoimmunity associated with WAS and may help to inform long-term therapeutic options.

Formononetin Inhibits Progression of Endometriosis via Regulation of p27, pSTAT3, and Progesterone Receptor: In Vitro and In Vivo Studies.

Formononetin is one of the phytoestrogens that functions like a selective estrogen receptor modulator (SERM). In this study, we evaluated the effects of formononetin on endometriosis progression in vitro and in vivo. After pathological confirmation, 10 eutopic and ectopic endometria were collected from patients with endometriosis. Ten eutopic endometria samples were collected from patients who did not have endometriosis. To determine the cytotoxic dose and therapeutic dose of formononetin, the concentration of 70% of the cells that survived after formononetin administration was estimated using a Cell counting kit-8 (CCK 8) assay. Western blot analysis was used to determine the relative expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 in the eutopic endometria without endometriosis, eutopic endometria with endometriosis, and ectopic endometria with endometriosis as the formononetin concentration was increased. We confirmed the effect of formononetin on apoptosis and migration in endometriosis using fluorescence-activated cell sorting (FACS) and wound healing assays, respectively. A mouse model of endometriosis was prepared using a non-surgical method, as previously described. The mice were intraperitoneally administered formononetin for four weeks after dividing them into control, low-dose formononetin (40 mg/kg/day) treatment, and high-dose (80 mg/kg/day) formononetin treatment groups. All the mice were euthanized after formononetin treatment. Endometriotic lesions were retrieved and confirmed using hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining of p27 was performed. We set the maximum concentration of formononetin administration to 80 μM through the CCK8 assay. Based on formononetin concentration, the expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 proteins were measured using Western blot analysis (N = 4 per group). The expression level of pERK, p27, and pSTAT3 in eutopic endometrium with endometriosis tended to decrease with increasing formononetin concentration, and a significant decrease was noted at 80 μM. The expression of p27 in ectopic endometrium with endometriosis was also significantly decreased at 80 μM of formononetin. FACS analysis revealed that formononetin did not significantly affect apoptosis. In the wound healing assay, formononetin treatment revealed a more significant decrease in the proliferation of the eutopic endometrium in patients with endometriosis than in the eutopic endometrium without endometriosis. Relative expression of sex hormone receptors decreased with increasing formononetin doses. Although no significant differences were observed in the ER, PR-A, ERβ/ERα, and PR-B/PR-A, significant down-regulation of PR-B expression was noted after formononetin treatment at 80 μM. In the in vivo study, endometriotic lesions in the formononetin-treated group significantly decreased compared to those in the control group. The relative expression of p27 using IHC was highest in the control group and lowest in the high-dose formononetin treatment group. Formononetin treatment was shown to inhibit the proliferation of eutopic and ectopic endometria in patients with endometriosis through the regulation of p27, pSTAT3, and PR-B. In an endometriosis mouse model, formononetin treatment significantly reduced the number of endometriotic lesions with decreased p27 expression. The results of this study suggest that formononetin may be used as a non-hormonal treatment option for endometriosis.

Perivascular localized cells commit erythropoiesis in PDGF-B-expressing solid tumors.

Tumors possess incessant growth features, and expansion of their masses demands sufficient oxygen supply by red blood cells (RBCs). In adult mammals, the bone marrow (BM) is the main organ regulating hematopoiesis with dedicated manners. Other than BM, extramedullary hematopoiesis is discovered in various pathophysiological settings. However, whether tumors can contribute to hematopoiesis is completely unknown. Accumulating evidence shows that, in the tumor microenvironment (TME), perivascular localized cells retain progenitor cell properties and can differentiate into other cells. Here, we sought to better understand whether and how perivascular localized pericytes in tumors manipulate hematopoiesis. To test if vascular cells can differentiate into RBCs, genome-wide expression profiling was performed using mouse-derived pericytes. Genetic tracing of perivascular localized cells employing NG2-CreERT2:R26R-tdTomato mouse strain was used to validate the findings in vivo. Fluorescence-activated cell sorting (FACS), single-cell sequencing, and colony formation assays were applied for biological studies. The production of erythroid differentiation-specific cytokine, erythropoietin (EPO), in TME was checked using quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA, magnetic-activated cell sorting and immunohistochemistry. To investigate BM function in tumor erythropoiesis, BM transplantation mouse models were employed. Genome-wide expression profiling showed that in response to platelet-derived growth factor subunit B (PDGF-B), neural/glial antigen 2 (NG2)+ perivascular localized cells exhibited hematopoietic stem and progenitor-like features and underwent differentiation towards the erythroid lineage. PDGF-B simultaneously targeted cancer-associated fibroblasts to produce high levels of EPO, a crucial hormone that necessitates erythropoiesis. FACS analysis using genetic tracing of NG2+ cells in tumors defined the perivascular localized cell-derived subpopulation of hematopoietic cells. Single-cell sequencing and colony formation assays validated the fact that, upon PDGF-B stimulation, NG2+ cells isolated from tumors acted as erythroblast progenitor cells, which were distinctive from the canonical BM hematopoietic stem cells. Our data provide a new concept of hematopoiesis within tumor tissues and novel mechanistic insights into perivascular localized cell-derived erythroid cells within TME. Targeting tumor hematopoiesis is a novel therapeutic concept for treating various cancers that may have profound impacts on cancer therapy.

Cellular and In Vivo Preclinical Pharmacodynamics and Pharmacology of Nipocalimab, an Anti-FcRn Blocking Therapeutic Antibody (P10-5.011)

<h3>Objective:</h3> To characterize the mechanism of action, pharmacodynamics and pharmacology of nipocalimab in preclinical models. <h3>Background:</h3> Nipocalimab is a fully human antibody being evaluated in over 10 autoantibody diseases including MG and CIDP. Nipocalimab binds with high affinity to the neonatal Fc receptor (FcRn) inhibiting IgG recycling and inducing autoantibody destruction while permitting key immune functions including IgG production. Here, the molecular, cellular and in vivo pharmacology of nipocalimab are shown to be well aligned with the pharmacodynamic and pharmacological effects observed in MG patients. <h3>Design/Methods:</h3> Nipocalimab in vitro receptor occupancy (RO) was assessed in a fluorescence-activated cell sorting assay in human aortic endothelial cells. In vivo, RO in circulating monocytes and total serum IgG were evaluated in human FcRn transgenic mice (Tg32 strain) or cynomolgus monkeys after i.v. infusions of up to 100 or 300 mg/kg nipocalimab, respectively. <h3>Results:</h3> Nipocalimab exposure at picomolar concentrations results in full RO within 30 minutes and inhibits IgG recycling in endothelial cells. In vivo, full RO was established within 2 hours in mice and 4 hours in monkeys and maintained at picomolar concentrations. The onset of IgG lowering due to inhibition of IgG recycling was detected within 1–2 days and was maintained during the period of full FcRn saturation. Duration of RO in peripheral blood monocytes in both preclinical species was dose-dependent, as was the duration of IgG clearance, suggesting a relationship between RO and maintenance of an increased IgG clearance rate. Nipocalimab in vitro FcRn RO and IgG recycling correlated well with in vivo RO and IgG lowering. <h3>Conclusions:</h3> Nipocalimab is a potent FcRn blocker inducing rapid and sustained lowering of IgG and IgG autoantibodies in preclinical studies. These results are consistent with findings from the phase 1 healthy volunteer (NCT02828046) and phase 2 VIVACITY study in gMG patients (NCT03772587). <b>Disclosure:</b> Dr. Ling has received personal compensation for serving as an employee of Janssen Pharmaceuticals (Johnson&amp;Johnson). Dr. Ling has stock in Janssen Pharmaceuticals (Johnson&amp;Johnson). Dr. Ling has received intellectual property interests from a discovery or technology relating to health care. Dr. Ling has received intellectual property interests from a discovery or technology relating to health care. Dr. Choudhury has stock in Janssen Pharmaceuticals. Mr. Tyler has received personal compensation for serving as an employee of AVROBIO, INC . Mr. Tyler has received personal compensation for serving as an employee of Orna Therapeutics. Mr. Tyler has received personal compensation for serving as an employee of Genevant Sciences. Dr. Avery has received personal compensation for serving as an employee of Lyndra Therapeutics. Dr. Avery has received personal compensation for serving as an employee of Xilio Therapeutics. Dr. Avery has received personal compensation for serving as an employee of Kisbee Therapeutics. Dr. Avery has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Momenta Pharmaceutical. Dr. Avery has received stock or an ownership interest from Life Biosciences.

Transverse myelitis following bivalent COVID‐19 booster vaccine and quadrivalent seasonal influenza vaccine

Transverse myelitis following bivalent <scp>COVID</scp>‐19 booster vaccine and quadrivalent seasonal influenza vaccine

CD142 promotes trophoblast cell migration by inhibiting BCL2-related autophagic degradation of IL-8.

The maintenance of migration of trophoblast cells is beneficial to pregnancy, and its weakening can lead to preeclampsia (PE). CD142 is considered as a classical motility-promoting factor. Our research aimed to explore the role of CD142 in trophoblast cell migration and potential mechanism. Through fluorescence-activated cell sorting (FACS) and gene transduction assays, CD142 expression levels of mouse trophoblast cell lines were upregulated and downregulated respectively. Then, the migratory level was detected through Transwell assays in different groups of trophoblast cells. The corresponding chemokines were screened by ELISA in different sorted trophoblast cells. Based on gene overexpression and knockdown assays, the production mode of identified valuable chemokine was analyzed by detecting gene and protein expression in trophoblast cells. Finally, the contribution of autophagy response to specific chemokine regulated by CD142 was explored by combining different groups of cells and autophagy regulators. Our results showed that both CD142 positive sorting and CD142 overexpression promoted the migratory ability of trophoblast cells, and trophoblast cells with the highest level of CD142 had the strongest migratory ability. In addition, CD142+ cells contained the highest level of IL-8. Consistently, CD142 overexpression promoted IL-8 protein expression in trophoblast cells while CD142 silencing was contrary. However, both CD142 overexpression and CD142 silencing did not affect IL-8 mRNA expression. Moreover, both CD142+ and CD142-overexpressed cells showed higher BCL2 protein expression and poorer autophagic activity. Importantly, autophagy activation with TAT-Beclin1 recovered the increased IL-8 protein expression in CD142+ cells. Obviously, the migratory ability of CD142+ cells inhibited by TAT-Beclin1 was recovered by the addition of IL-8 recombinant factor. In conclusion, CD142 inhibits the degradation of IL-8 through the inhibition of BCL2-Beclin1-autophagy signal transduction, thereby promoting the migration of trophoblast cells.

Anethole attenuates lung cancer progression by regulating the proliferation and apoptosis through AKT and STAT3 signaling

Background: The main constituent of fennel, anethole, exerts beneficial effects in many diseases, including antiproliferative and tumoricidal capacities. This study aimed to investigate the effect of anethole on the human non-small cell lung cancer (NSCLC) cell line, A549, and the related molecular mechanisms.&#x0D; Methods: The proliferation was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Apoptosis situation was determined using fluorescence-activated cell sorting and DNA fragmentation assay combined with immunoblotting assay probing-related protein expression. Phosphoinositide 3-kinases-protein kinase B (PI3K-AKT) and signal transducer and activator of transcription 3 (STAT3) signaling proteins were evaluated using immunoblotting. Xenograft model was constructed to determine the tumor volume and weight and to evaluate the expression of Ki67 via immunohistochemistry and cleaved caspase 3 via immunoblotting.&#x0D; Results: Anethole inhibited proliferation and clonal growth of A549 cells. The promotion of A549 cell apoptosis by anethole was evidenced by increased apoptotic ratio, abundant DNA fragments, and caspase-3 activation. Key proteins in PI3K-AKT and STAT3 signaling pathways were decreased in anethole group. Additionally, xenografted tumors in anethole group retarded with decreased Ki67 and increased cleaved caspase 3 expression.&#x0D; Conclusions: Anethole induced tumor cell apoptosis and retarded cell proliferation. AKT and STAT3 signaling pathways might be involved in the effects of anethole. Our findings highlight the feasibility of anethole in the treatment of lung cancer.

Anethole attenuates lung cancer progression by regulating the proliferation and apoptosis through AKT and STAT3 signaling

Background: The main constituent of fennel, anethole, exerts beneficial effects in many diseases, including antiproliferative and tumoricidal capacities. This study aimed to investigate the effect of anethole on the human non-small cell lung cancer (NSCLC) cell line, A549, and the related molecular mechanisms.&#x0D; Methods: The proliferation was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Apoptosis situation was determined using fluorescence-activated cell sorting and DNA fragmentation assay combined with immunoblotting assay probing-related protein expression. Phosphoinositide 3-kinases-protein kinase B (PI3K-AKT) and signal transducer and activator of transcription 3 (STAT3) signaling proteins were evaluated using immunoblotting. Xenograft model was constructed to determine the tumor volume and weight and to evaluate the expression of Ki67 via immunohistochemistry and cleaved caspase 3 via immunoblotting.&#x0D; Results: Anethole inhibited proliferation and clonal growth of A549 cells. The promotion of A549 cell apoptosis by anethole was evidenced by increased apoptotic ratio, abundant DNA fragments, and caspase-3 activation. Key proteins in PI3K-AKT and STAT3 signaling pathways were decreased in anethole group. Additionally, xenografted tumors in anethole group retarded with decreased Ki67 and increased cleaved caspase 3 expression.&#x0D; Conclusions: Anethole induced tumor cell apoptosis and retarded cell proliferation. AKT and STAT3 signaling pathways might be involved in the effects of anethole. Our findings highlight the feasibility of anethole in the treatment of lung cancer.

Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) as a novel presentation of CNS autoimmunity in a pediatric patient with Wiskott-Aldrich syndrome (WAS)

Objective Report a novel case of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) presenting as relapsing bilateral optic neuritis in a pediatric patient with Wiskott-Aldrich syndrome (WAS). Background WAS is a rare X-linked primary immunodeficiency caused by mutations in the WAS gene that leads to increased susceptibility to infections, thrombocytopenia, eczema, malignancies, and autoimmunity. Known CNS autoimmune manifestations include cerebral vasculitis, but optic neuritis, CNS demyelination, and MOGAD have not been previously reported. Design/Methods Chart review Results A 5-year-old boy with a history of chronic immune thrombocytopenia, hypogammaglobulinemia, anemia, and focal epilepsy developed binocular vision loss. MRI of the brain demonstrated enlargement of bilateral optic nerves with marked enhancement of the nerve sheaths consistent with optic neuritis, as well as multiple small enhancing supratentorial lesions. He was treated with pulse methylprednisolone followed by oral steroid taper, and he returned to baseline with no reported residual visual deficits. Five months later, he experienced a relapse of bilateral vision loss, and repeat MRI re-demonstrated bilateral optic neuritis as well as resolution of prior brain lesions. He was treated with repeat course of steroids and experienced moderate improvement in his vision. Rituximab was then initiated to prevent further relapses of optic neuritis while treating his chronic suspected immune-mediated thrombocytopenia. Myelin oligodendrocyte glycoprotein antibody (MOG-IgG) via serum fluorescence-activated cell sorting assay was positive (titer 1:100), confirming a diagnosis of MOGAD. At age six, molecular panel testing for genes associated with primary immunodeficiency identified a missense WAS gene variant. He was subsequently found to have decreased WAS protein expression consistent with a diagnosis of WAS. Conclusions We describe a case of pediatric MOGAD presenting with multiphasic bilateral optic neuritis in a patient with WAS. This case expands the reported spectrum of CNS autoimmunity associated with WAS and may help to inform indications for therapeutic options such as bone marrow transplant.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1β [IL-1β], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409’s efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion

ObjectivesPancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion. MethodsTo identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen. ResultsWe identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes. ConclusionsWe demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion.

Positive Predictive Value of MOG-IgG for Clinically Defined MOG-AD Within a Real-World Cohort.

Myelin oligodendrocyte glycoprotein antibody associated disease (MOG-AD) is a CNS demyelinating disease, typically presenting with optic neuritis, transverse myelitis, and/or ADEM-like syndromes. The positive predictive value (PPV) of MOG-IgG testing by live cell-based assay was reported to be 72% in a study performed at the Mayo Clinic using a cut-off of 1:20. PPV may vary depending upon the tested population, thus supporting further investigation of MOG-IgG testing at other centers. In this real-world institutional cohort study, we determined the PPV of serum MOG-IgG for clinically defined MOG-AD in our patient population. The Massachusetts General Brigham Research Patient Data Registry database was queried for patients with positive serum MOG-IgG detection, at least once, between January 1, 2017 and March 25, 2021. All were tested via the MOG-IgG1 fluorescence-activated cell sorting assay (Mayo Laboratories, Rochester, MN). MOG-IgG positive cases were reviewed for fulfillment of typical MOG-AD clinical features, determined by treating neurologists and study authors. Of 1,877 patients tested, 78 (4.2%) patients tested positive for MOG-IgG with titer ≥1:20, and of these, 67 had validated MOG-AD yielding a PPV of 85.9%. Using a ≥1:40 titer cutoff, 65 (3.5%) tested positive and PPV was 93.8%. Three MOG positive cases had a prototypical multiple sclerosis diagnosis (RRMS n = 2, titers 1:20 and 1:40; PPMS n = 1; 1:100). The treating diagnosis for one RRMS patient with a 1:40 titer was subsequently modified to MOG-AD by treating neurologists. Validated diagnoses of the remaining positive patients without MOG-AD included: migraine (n = 2, titers 1:20, 1:100), inclusion body myositis (n = 1, titer 1:100), autoimmune encephalitis (n = 2, titers 1:20, 1:20), hypoxic ischemic brain injury (n = 1, titer 1:20), IgG4-related disease (n = 1, titer 1:20), and idiopathic hypertrophic pachymeningitis (n = 1, titer 1:20). In our cohort, the PPV for MOG-IgG improved utilizing a titer cut-off of ≥1:40. The presence of positive cases with and without demyelinating features, emphasizes a need for testing in the appropriate clinical context, analysis of titer value and clinical interpretation.

Abstract 6008: Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICIs) have generated durable clinical responses in multiple tumor types, but their therapeutic efficacy is limited by development of primary or acquired resistance. The tumor microenvironment (TME) contains many factors influencing ICI responses. The ratio of effector T-cells (Teffs) and regulatory T-cells (Tregs) in tumors has been shown to be associated with the efficacy of ICIs in murine models, indicating that increasing Teffs or decreasing Tregs in tumor may prevent development of resistance to immunotherapy. There is a high and restricted expression of CC motif chemokine receptor 8 (CCR8) on tumor-infiltrating Tregs, whereas they are negligibly expressed on Teffs. Deletion of tumor-infiltrating Tregs via murine anti-CCR8 surrogates has shown effective anti-tumor activity in tumor models.LM-108 is a novel humanized anti-CCR8 monoclonal antibody with engineered IgG1 that enhances antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of CCR8 and Foxp3 on tumor-infiltrating lymphocytes (TILs) was analyzed using published single-cell sequencing data. The binding affinity of LM-108 was assessed by flow cytometry using CCR8 high- and low-expressing cell lines. In vitro measurements of ADCC and antibody-dependent cellular phagocytosis (ADCP) mediated by LM-108 were conducted in CCR8 overexpressing HEK293, CHO-K1, and Jurkat cells. Furthermore, in vivo anti-tumor activity of LM-108 as monotherapy and in combination with anti-PD-1 therapy was evaluated in syngeneic models and PD-1-resistant tumor models with human CCR8 knock-in mice. Analysis of published single-cell sequencing data showed that in comparison with CCR4, CCR8 gene expression was specifically overlapped with Foxp3 in TILs of breast cancer. Fluorescence-activated cell sorting (FACS) assay revealed that LM-108 specifically binds with high affinity to human CCR8 in a dose-dependent manner. ADCC and ADCP effects induced by LM-108 were observed in HEK293 and CHO-K1 cells with different levels of CCR8 expression. In vivo data of CT26 syngeneic model showed that LM-108 murine surrogate antibody alone or in combination with anti-PD-1 antibody significantly inhibited tumor growth. Treatment with LM-108 effectively depleted tumor-infiltrating Tregs accompanied by increase in CD8/Treg ratio in tumors, leading to significant anti-tumor activity in MC38 model. Finally, LM-108 showed a synergistic therapeutic efficacy in PD-1 resistant models in combination with anti-PD-1 antibody. To conclude, LM-108 is a novel Fc-optimized CCR8 antibody that selectively depletes tumor infiltrating Tregs thereby improving anti-tumor immune response either as monotherapy or combination therapy. Thus, LM-108 can be a promising therapeutic approach to overcome ICI resistance in cancer patients. Citation Format: Jie Luo, Wentao Huang, Junwei Yang, Jin Li, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6008.

HMT Exerts an Anticancer Effect by Targeting PAK-1

Heamatang (HMT) is a classic medicinal formula used in traditional Chinese and Korean medicine; it contains seven distinct components, mainly of herbal origin. HMT is used as an antiaging remedy, treating urinary disorders and increasing energy and vitality. However, the therapeutic applications of this formula have not been evaluated with evidence-based science. Therefore, we assessed HMT through various in vitro methods, including cell viability assay, fluorescence-activated cell sorting assay (FACS), Western blotting, migration assay, three-dimensional (3D) cell culture, siRNA-mediated PAK-1 knockdown, and crystal violet assays. HMT decreased PAK-1 expression in PC-3 cells and inhibited cell viability, growth, and motility. The inhibition of cell motility by HMT was correlated with PAK-1-mediated inhibition of Lim domain kinase (LIMK) and cofilin. HMT induced G1 arrest and apoptosis through the transcriptional regulation of cell cycle regulatory proteins and apoptosis-related proteins (increase in c-cas3 and inhibition of PARP and BCL-2). Moreover, HMT suppressed PAK-1 expression, leading to the inhibition of AKT activities. Finally, we showed that decursin was the active ingredient involved in the inhibitory effect of HMT on PAK-1. Our findings demonstrated that HMT exerts its anticancer influence through the inhibition of PAK-1. The HMT formula could be applied in various fields, including functional health food and pharmaceutical development.

Luteolin promotes macrophage-mediated phagocytosis by inhibiting CD47 pyroglutamation

‘Don't eat me’ signal of CD47 is activated via its interaction with SIRPα protein on myeloid cells, especially phagocytic cells, and prevents malignant cells from anti-tumor immunity in which pyroglutamate modification of CD47 by glutaminyl-peptide cyclotransferase-like protein (isoQC) takes an important part evidenced by our previous report that isoQC is an essential regulator for CD47-SIRPα axis with a strong inhibition on macrophage-mediated phagoctyosis. Therefore, we screened for potential isoQC inhibitors by fluorescence-activated cell sorting assay and identified luteolin as a potent compound that blocked the pyroglutamation of CD47 by isoQC. We further demonstrated that luteolin directly bound to isoQC using pull-down assay and isothermal calorimetric (ITC) assay. In consistency, we showed that luteolin markedly abrogated the cell-surface interaction between CD47 and SIRPα in multiple myeloma H929 cells and consequently promoted the macrophage-mediated phagocytosis. Collectively, our study discovered a promising lead compound targeting isoQC, luteolin, which functions distinctly from current CD47 antibody-based drugs and therefore may potentially overcome the clinical side effects associated with CD47 antibody treatment-induced anemia.