Carboxyfluorescein Succinimidyl Ester Research Articles

P10.01.A A PATIENT-DERIVEDEX VIVO ORGANOID MODEL ALLOWS EVALUATION OF TUMOR TREATING FIELDS (TTFIELDS) EFFECTS ON GLIOBLASTOMA, ANAPLASTIC EPENDYMOMA AND OLIGODENDROGLIOMA SAMPLES

Abstract BACKGROUND The use of TTFields, which are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), has shown its clinical efficacy in the treatment of newly diagnosed glioblastoma (GBM) patients at 200 kHz. Nevertheless, more knowledge is required for the prediction of individual tumors’ responsiveness to treatment. Recently, we developed patient-derived three-dimensional GBM tissue culture models to screen patient tumors for TTFields treatment susceptibility. Here, we extended these models to brain tumors beyond GBM, namely anaplastic ependymoma (AE) and oligodendroglioma. We cultured tumor-organoids as microtumors on murine organotypic hippocampal slice cultures (OHSCs) and subjected them to TTFields at 200 kHz. MATERIAL AND METHODS We prepared OHSCs by slicing the brains of mice 5-8 days postpartum to a thickness of 350 µm using a vibratome and culturing them for 14 days. We obtained organoids from freshly resected tumor tissue from three GBM, one AE, and one oligodendroglioma patient, each. The organoids were cultured for 14 days as well and then stained with carboxyfluorescein succinimidyl ester. Next, we placed the organoids onto the OHSCs and administered TTFields at 200 kHz and 1.5 V/cm for 72 h using the inovitro™ laboratory research system. We quantified microtumor growth by fluorescence imaging. RESULTS The three tumor entities, in particular organoids from GBM, AE and oligodendroglioma, grew as microtumors after being placed onto OHSCs. TTFields treatment at 200 kHz led to significantly decreased microtumor size of the three analysed GBM samples as well as the AE sample. However, the oligodendroglioma microtumor size was not significantly reduced. CONCLUSION Based on clinical experience, we expected that TTFields lead to a reduction in microtumor size. However, to our knowledge, we were the first to apply TTFields to patient-derived AE and oligodendroglioma samples. The lack of TTFields-effects on the latter might be due to different inter-patient sensitivity or suboptimal TTFields frequency. Nevertheless, our work is proof of principle that an organoid-based patient-derived 3D tissue culture model could be used to evaluate effects of TTFields on different brain tumor entities.

Identification, sorting and profiling of functional killer cells via the capture of fluorescent target-cell lysate.

Assays for assessing cell-mediated cytotoxicity are largely target-cell-centric and cannot identify and isolate subpopulations of cytotoxic effector cells. Here we describe an assay compatible with flow cytometry for the accurate identification and sorting of functional killer-cell subpopulations in co-cultures. The assay, which we named PAINTKiller (for 'proximity affinity intracellular transfer identification of killer cells'), relies on the detection of an intracellular fluorescent protein 'painted' by a lysed cell on the surface of the lysing cytotoxic cell (specifically, on cell lysis the intracellular fluorescein derivative carboxyfluorescein succinimidyl ester is captured on the surface of the natural killer cell by an antibody for anti-fluorescein isothiocyanate linked to an antibody for the pan-leucocyte surface receptor CD45). The assay can be integrated with single-cell RNA sequencing for the analysis of molecular pathways associated with cell cytotoxicity and may be used to uncover correlates of functional immune responses.

Comparing [3H] thymidine LPT and CFSE assay to assess lymphocyte proliferation in beryllium-exposed sarcoidosis patients

The detection of antigen specific lymphocyte responses plays a vital role in the diagnosis of various diseases. Beryllium-specific [3H] thymidine lymphocyte proliferation test (LPT) is regarded as a gold standard in identifying chronic beryllium disease (CBD) cases. Alternatively, flow cytometric based carboxyfluorescein succinimidyl ester (CFSE) assay, has several benefits as opposed to LPT, since it further permits both phenotypical characterization and functional analysis of proliferating lymphocyte subsets.The suitability of both LPT and CFSE assay to therefore detect beryllium sensitivity in a group of Be-exposed sarcoidosis patients with suspected beryllium exposure, was evaluated in this study. The clinical relevance of the test responses, expressed as stimulation indices (SI), were additionally compared on a group and individual level.Agreement in clinical interpretation of the test responses between both methods was observed in 4 out of 5 recruited patients, when considering total lymphocyte population i.e., CD3+ and CD19+-cells combined, on day 7 and with CFSE-SI >1.5, when compared with LPT-SI >2.5. Variability in responses to beryllium was additionally evaluated in Be-exposed sarcoidosis patients and compared with healthy controls.To conclude, both LPT and CFSE assay are suitable assays to detect Be sensitivity in Be-exposed sarcoidosis patients. At the same time, flow cytometric based CFSE assay has the edge over LPT in identifying the relevant proliferating lymphocyte populations. As such, when comparing two or more methods, factors that contribute to assay variability such as timepoints, lymphocyte subsets and number of replicates should always be accounted for.

Macrophages polarize to the pro-inflammatory phenotype and delay neutrophil efferocytosis to augment Aggregatibacter actinomycetemcomitance JP2 clearance.

The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis. Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood. Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model. Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results. As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage.

Obstructive Sleep Apnea-Induced Hypertension Is Associated With Increased Gut and Neuroinflammation.

Background Obstructive sleep apnea (OSA) is an independent risk factor for the development of hypertension. We have demonstrated that OSA induces gut dysbiosis, and this dysbiotic microbiota contributes to hypertension. However, the mechanisms linking gut dysbiosis to blood pressure regulation remain unclear. Recent studies demonstrate that gut dysbiosis can induce a proinflammatory response of the host resulting in peripheral and neuroinflammation, key factors in the development of hypertension. We hypothesized that OSA induces inflammation in the gut that contributes to neuroinflammation and hypertension. Methods and Results OSA was induced in 8-week-old male rats. After 2 weeks of apneas, lymphocytes were isolated from aorta, brain, cecum, ileum, mesenteric lymph node, and spleen for flow cytometry. To examine the role of interleukin-17a, a monoclonal antibody was administered to neutralize interleukin-17a. Lymphocytes originating from the gut were tracked by labeling with carboxyfluorescein succinimidyl ester dye. OSA led to a significant decrease in T regulatory cells along with an increase in T helper (TH) 17 cells in the ileum, cecum, and brain. Interleukin-17a neutralization significantly reduced blood pressure, increased T regulatory cells, and decreased TH1 cells in the ileum, cecum, and brain of OSA rats. TH1, TH2, and TH17 cells from the gut were found to migrate to the mesenteric lymph node, spleen, and brain with increased frequency in rats with OSA. Conclusions OSA induces a proinflammatory response in the gut and brain that involves interleukin-17a signaling. Gut dysbiosis may serve as the trigger for gut and neuroinflammation, and treatments to prevent or reverse gut dysbiosis may prove useful in reducing neuroinflammation and hypertension.

RSV-specific T cell epitope presentation in the respiratory tract differs between adults and neonates as revealed by a novel T cell proliferation assay

Abstract Respiratory syncytial virus (RSV) invades the respiratory epithelium and provokes severe symptoms in young children leading to infant mortality. CD8 tissue resident memory (TRM) cells are associated with protection in adults against RSV, but are poorly established in neonatal mice consequent to primary infection. We hypothesized that antigen presentation contributes to age-dependent TRM establishment, thus we sought to define antigen kinetics in RSV-infected adult and neonatal mice. CB6F1/J adult and neonatal mice were infected with RSV and lung tissue were isolated over the course of acute and convalescent infection. Single cell lung suspension was co-cultured in vitrowith RSV K dM2 82–90(M2) or D bM 187–195(M) epitope-specific T cells isolated from CD8 T cell receptor (TCR) transgenic mice. T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) to quantify proliferative response 48 hours post-incubation at 37°C via flow cytometry. From lung tissue isolated at 7 days post infection (dpi), M and M2-specific CD8 T cells proliferated in response to co-culture; however, higher rates were induced by neonatal lung tissue primarily by M-specific T cells. By 8 and 9 dpi, proliferation induced by isolated neonatal lung continued while proliferation induced by adult lung tissue was not quantifiable by our assay. Our findings suggest antigen availability in neonates exceeds that of adults and is age and epitope-dependent. These discoveries indicate that antigen accessibility does not independentlycorrespond with the magnitude of the T cell response in early life. Exploring the inflammatory contributions to RSV-specific T cell defenses is required as this may aid in vaccine design and understanding disease pathogenesis. Supported by grant from NIH R01-AI154619.

MP14-05 EXTRACELLULAR VESICLE BIOMARKER SIGNATURE DETECTION PLATFORM FOR GUIDING CANCER DETECTION AND THERAPY

You have accessJournal of UrologyCME1 Apr 2023MP14-05 EXTRACELLULAR VESICLE BIOMARKER SIGNATURE DETECTION PLATFORM FOR GUIDING CANCER DETECTION AND THERAPY Thomas Osinski, Samuel Walker, Karen Doersch, Jonathan Flax, and James McGrath Thomas OsinskiThomas Osinski More articles by this author , Samuel WalkerSamuel Walker More articles by this author , Karen DoerschKaren Doersch More articles by this author , Jonathan FlaxJonathan Flax More articles by this author , and James McGrathJames McGrath More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular Vesicles (EVs) have tremendous potential in cancer diagnostics and personalized cancer therapeutics. They are abundant in all biofluids, carry molecular markers of their cell-of-origin and can be readily obtained via noninvasive techniques. Current EV biomarker assessments have several limitiations. Pooled EVs from samples have low detection sensitivity for less abundant biomarkers and preclude colocalization of biomarkers on individual EVs. Currently, expensive specialized devices requiring large biofluid inputs are needed for more detailed EV analysis. Here, we develop an easy to use and inexpensive device that permits optical detection of multiple markers on individual EVs using only 10 µl of serum. This work investigates whether the microfluidic strategy we call ‘catch and display for liquid biopsy [CAD-LB]’ can detect biomarkers on single EVs. We also sought to determine the dynamic range of accurate quantitation using purified EVs and serum and validate detection of immune checkpoint proteins (PD-L1 and/or PD-1), which may help to distinguish immune checkpoint immunotherapy (ICI) responders from non-responders in bladder cancer. METHODS: For this study EVs were derived from: 1. ultracentrifuge purified urinary EVs; 2. EVs from media of 5637 bladder cancer and primary bladder epithelium cell lines and; 3. Serum from patients with advanced urothelial carcinoma or healthy controls. EVs were labeled with the pan-EV marker carboxyfluorescein succinimidyl ester and fluorescently labeled primary antibodies. Small volumes (10 µL) of solutions were captured on our membrane with nanopores in a transparent membrane and imaged using fluorescence microscopy. Detection range was assessed by serial dilutions of EVs. EV surface antigen labeling was assessed by CAD-LB. RESULTS: 1. Counting curves demonstrate a wide dynamic range of CAD-LB (102 – 106 EVs) for purified EVs while serum plateaus at lower inputs (∼104 EVs). 2. CAD-LB detects PD-1 and PD-L1 biomarkers in urothelial carcinoma patients’ serum. 3. CAD-LB detects T cell biomarker CD3 on approximately 1-2% of EVs in healthy control serum. CONCLUSIONS: CAD-LB can detect rare and clinically significant EV associated biomarkers in conditioned medium and patient serum samples. Furthermore, CAD-LB provides single EV characterization with small sample volumes. These qualities make CAD-LB a potentially useful tool to help discover novel markers that could aid in diagnosis and treatment decisions for cancer patients. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e183 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Thomas Osinski More articles by this author Samuel Walker More articles by this author Karen Doersch More articles by this author Jonathan Flax More articles by this author James McGrath More articles by this author Expand All Advertisement PDF downloadLoading ...

Testosterone Propionate Promotes Proliferation and Viability of Bone Marrow Mesenchymal Stem Cells while Preserving Their Characteristics and Inducing Their Anti-Cancer Efficacy

Various studies have reported the effects of testosterone on different cell types, yet bone marrow-derived mesenchymal stem cells’ cellular responses to testosterone remain unknown. To investigate the effects of testosterone propionate, an oil-soluble short-acting form of testosterone, on human bone marrow-derived mesenchymal stem cells’ proliferation and viability after 24 hours of incubation. We also investigated the impact of testosterone propionate on bone marrow-derived mesenchymal stem cell’s polarization and cytotoxicity on K562 leukemia cell line. In vitro study. We expanded commercially available bone marrow derived mesenchymal stem cells in vitro and treated them with testosterone propionate at concentrations ranging from 10-6-10-10 M for 24 hours. Ideal concentration was determined by evaluating cellular viability and proliferation with Annexin V/Propidium Iodide assay and carboxyfluorescein succinimidyl ester staining. The characteristic features of bone marrow-derived mesenchymal stem cells were evaluated by immunophenotyping and investigating their differentiation capacities. Bone marrow-derived mesenchymal stem cells’ cytotoxic properties upon testosterone propionate treatment were determined by co-culturing the cells with K562 cells and with confocal imaging investigating polarization. Testosterone propionate promoted proliferation and maintained the viability of bone marrow-derived mesenchymal stem at 10-8 M concentration. Further evaluations were conducted with the determined dose. The results showed that, apart from promoting mesenchymal stem cells’ polarization and increasing their cytotoxicity on K562 cells, testosterone propionate did not alter differentiation capacities of bone marrow-derived mesenchymal stem cells and certain cell surface markers, but led to a significant increase in HLA-DR expression. The findings reveal that testosterone propionate promotes the proliferation and survival of bone marrow-derived mesenchymal stem cells in a dose-dependent manner without hampering their differentiation capacities, induces their polarization to the pro-inflammatory phenotype, and increases their cytotoxicity on the K562 cell line.

Individual variation in and lateral asymmetry of mouse epididymal draining lymph nodes.

Draining lymph nodes (LNs) are pivotal sites for maintaining tolerance to self-antigens as well as eliciting immune responses to exogenous antigens. The epididymis is a male reproductive organ with a unique local immune environment. Although mice are the most commonly used laboratory animals for immunology research, there are no detailed descriptions of the anatomical location and function of LNs that drain the epididymis. Evans blue labeling was utilized to explore lymphatic drainage of the epididymis in eight- to ten-week-old male C57BL/6 mice. We confirmed the lymphatic drainage of the epididymis in mice using the objective technique of carboxyfluorescein succinimidyl ester (CFSE)-labeled cells. By combined Evans blue labeling and fluorescent labeling, we found that 1) the patterns of epididymal LN drainage are highly heterogeneous between individual mice; 2) the leftside LNs participate in drainage more frequently than the right-side LNs; and 3) epididymal lymphatic drainage bypasses both the paraaortic and renal LNs in some mice. These data highlighted the need to consider the individual variation in and lateral asymmetry of draining LNs when characterizing the regional immunology of the mouse epididymis.

S-57-6: OBSTRUCTIVE SLEEP APNEA INDUCED GUT DYSBIOSIS STIMULATES A PROINFLAMMATORY RESPONSE THAT PROMOTES NEUROINFLAMMATION AND HYPERTENSION

Objective: Obstructive sleep apnea (OSA) is an independent risk factor for hypertension (HT). We have demonstrated that OSA induces gut dysbiosis, and a dysbiotic microbiota is causal in the development of HT. The mechanisms linking gut dysbiosis to blood pressure (BP) regulation are unclear. Recent studies demonstrate gut dysbiosis induces a pro-inflammatory host response resulting in peripheral- and neuro-inflammation, key factors in the development of HT. We hypothesized that OSA induced gut dysbiosis elicits a pro-inflammatory response that promotes neuroinflammation and HT. Methods: OSA was induced in 8-week-old male rats by repeatedly inflating a surgically implanted tracheal balloon. Sham rats underwent balloon implantation without inflations. After two weeks of apneas, lymphocytes were isolated from aorta, brain, cecum, ileum, mesenteric lymph node, and spleen for assessment by flow cytometry. In studies examining IL-17, a monoclonal antibody to neutralize IL-17 was injected every 48 hours during the 2 weeks of OSA. To track lymphocytes originating from the gut, cells in the Peyers patches of the small intestine were labelled by injection of carboxyfluorescein succinimidyl ester (CFSE) dye. Results: Two weeks of OSA significantly decreased anti-inflammatory regulatory T (Treg) cells and increased TH17 (IL-17+) in the brain, cecum, and ileum (n = 7, p < 0.05). A significant increase in the Th17/Treg ratio was observed in brain, cecum and ileum of OSA, versus sham, rats (n = 7, p < 0.05). To examine the role of TH17 cells, we injected an IL-17 neutralizing antibody or control IgG antibody during the 2 weeks of OSA. Compared to OSA rats receiving IgG control, neutralization of IL-17 significantly reduced BP of OSA rats (n = 6, p < 0.05). Additionally, neutralization of IL-17 resulted in a significant increase of Tregs and decreased Th1 cells in brain, cecum and ileum of OSA rats (n = 6, p < 0.01). To examine the distribution of lymphocytes originating from the gut, cells in the Peyers patches of sham and OSA rats were labelled with CFSE. We observed significant increases in CFSE+ Th1, Th2, and Th17 cells in the brain, MLN and spleen of OSA as compared to sham rats (n = 6, p < 0.05). Conclusion: Overall, we found that OSA induced gut dysbiosis is associated with a pro-inflammatory response in the gut and brain that involves IL-17 signaling. Our findings suggest that gut dysbiosis may serve as the trigger for widespread inflammation, and treatment strategies to prevent or reverse gut dysbiosis may prove useful in reducing neuroinflammation and HT.

Anti-leukemic activity of Cyperus rotundus L. on human acute myeloid leukemia HL-60 cells in vitro

Context: Acute myeloid leukemia (AML) is the most common form of acute leukemia. Currently, many people use medicinal herbs for the treatment of cancers. Nut grass (Cyperus rotundus L.) is a medicinal plant widely used in conventional medicine due to its role as an anti-cancer. Aims: To evaluate the effect of Cyperus rotundus tuber (CRT) ethanolic extract on cell proliferation, differentiation, cell cycle, and apoptotic of HL-60 cells. Methods: HL-60 cells line as a model for AML was cultured under the influence of CRT extract concentrations (35.4, 354, and 3540 µg/mL) for 48 h. Furthermore, the cell was subjected to carboxyfluorescein succinimidyl ester staining for proliferation, using a CD117 marker for differentiation. Annexin V-FITC and PI for cell cycle and apoptosis. The effect of this herb was studied by flow cytometry. The data were statistically analyzed with one-way ANOVA (p≤0.05) and the Tukey test using SPSS version 16 for Windows. Results: The results showed that the CRT inhibited proliferation activity, differentiation, cell cycle arrest, and apoptosis. Conclusions: The data clearly showed the potential anti-cancer activity of CRT on HL-60 cells and suggested that it could help develop promising therapeutic agents for AML treatments.

Cytotoxicity of Human Hepatic Intrasinusoidal Gamma/Delta T Cells Depends on Phospho-antigen and NK Receptor Signaling.

We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells. γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo. The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1. The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

The AP-1/ETS Transcription Factor Network Regulates Stem Cell Quiescence and Chemotherapy Resistance in Acute Myeloid Leukemia

Despite of the continuous improvements in the treatment for acute myeloid leukemia (AML), therapy resistance and disease relapse have been persistent and attributed to leukemia stem cells (LSCs). Recent single cell RNA sequencing (Galen et al, Bernstein 2019) and deconvoluted bulk RNA sequencing analysis (Zeng et al, Dick 2022) show hierarchical cellular organization in individual AML patient specimens, with LSCs, and quiescent LSCs in particular, at its apex. However, the mechanisms controlling LSCs and their quiescence remain elusive, hindering the development of effective targeted therapies. Here, using human patient AML specimens, we show that activator protein 1 (AP-1) and E26 transformation-specific (ETS) family transcription factors (TFs) coordinately regulate LSC quiescence and disease persistence after chemotherapy. A chemical label tracing technique using carboxyfluorescein succinimidyl ester (CFSE) has emerged as a powerful tool to identify quiescent LSCs, and using this technique, we and others have demonstrated that CFSE-label retaining quiescent LSCs isolated from patient leukemia cells exhibit potent leukemia initiating property, resistance to conventional chemotherapy, and epigenetic plasticity (Takao et al, Kentsis 2018 ASH, Ebinger et al, Jeremias 2016 and 2020). Here, we combined this method with functional genomic approaches to elucidate the mechanisms controlling quiescent LSCs and identify novel therapeutic targets to overcome chemotherapy resistance. First, to identify the candidate genes for LSC quiescence regulators, we performed gene expression and chromatin accessibility profiling of label-retaining quiescent LSCs isolated from human patient leukemia cell-engrafted NSG mice in diverse subtypes of AMLs using mRNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). We identified that several transcription factors important for normal hematopoiesis, which included AP-1 TFs, such as JUN and JUNB, and ETS TFs, such as ETS1 and FLI1, were differentially expressed in quiescent LSCs. Chromatin accessibility profiling further revealed the predominant occupancy of AP-1 and ETS TF family binding motifs in differentially accessible chromatin loci in quiescent LSCs. Importantly, gene expression signatures detected in quiescent LSCs showed significant similarity to gene expression profiles of residual leukemia cells observed in chemotherapy-treated AML patients (GSE116256, Galen et al. 2019), supporting the clinical relevance of our models. Next, to develop a functional screen system to identify the regulators of quiescent LSCs in human patient AML-bearing mice, we designed a lentiviral vector encoding mCherry fluorescent protein and doxycycline-inducible cDNAs marked by specific barcode sequences. cDNA library screening using this lentiviral vector identified several TFs as regulators for AML LSC quiescence, which included JUN as a positive regulator, and ETS1 and FLI1 as negative regulators (Figure 1). Finally, given the evidence of transcriptional activity of JUN to regulate LSC quiescence, we hypothesized that JUN activity may be also associated with chemotherapy resistance and disease persistence in patients. To investigate this, we analyzed JUN expression in individual AML cells in diverse patients before and after chemotherapy treatment (GSE116256, Galen et al. 2019). We found JUN to be highly expressed in persistent AML cells upon induction chemotherapy, especially in leukemia cells with features of hematopoietic stem cells (HSC-like), while ETS1 and FLI1 expression was not (Figure 2). These results argue that JUN activity may be required for chemotherapy resistance, LSC persistence, and ultimate AML relapse. In all, human AML LSC quiescence and therapy response can be dynamically regulated by a distinct transcription factor network. This work defines molecular mechanisms controlling AML LSC quiescence. We anticipate that these findings will lead to the elucidation of essential properties of LSC quiescence and the design of therapeutic strategies for their clinical identification and control. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dimethyl Fumarate Inhibits Fibroblast Like Synoviocytes-mediated Inflammation and Joint Destruction in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) as a chronic inflammatory disorder affects around 1% of the world population. Fibroblast-like synoviocyte (FLS), one of the main cells in RA pathogenesis is characterized by hyperproliferation and resistance to apoptosis resulting to synovial hyperplasia. Dimethyl fumarate (DMF) has been licensed for the treatment of multiple sclerosis (MS) and psoriasis; however, its role in RA is unknown. DMF has immunomodulatory properties and may be considered as therapeutic approach in RA treatment. In this study, we aimed to investigate the effect of DMF on controlling FLS-mediated synovial inflammation and joint destruction in RA. FLSs were isolated from synovial tissues of 8 patients with RA and treated with DMF. Apoptosis rate was analyzed by Annexin V-FITC. Cell proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) dye. The matrix metalloproteinase 3 (MMP3) and NF-кB pathway protein (p65) mRNA expression were evaluated by RT-PCR. Also, the IL-6 production and lactate release were measured in FLS supernatant. DMF treatment decreased the cell proliferation and increased apoptosis in a dose dependent manner. DMF-treated FLS showed a reduction in IL-6 and lactate release. Moreover, it was revealed that DMF inhibited the expression of p65 and MMP3. Our data demonstrate that DMF treatment suppresses the aggressive and inflammatory features of RA FLSs. Our Results suggest that DMF might be expected to be evaluated as a therapy for RA.

The combination of Elephantopus scaber and Phaleria macrocarpa leaves extract promotes anticancer activity via downregulation of ER-α, Nrf2 and PI3K/AKT/mTOR pathway

BackgroundElephantopus scaber and Phaleria macrocarpa have recently been interested as novel anticancer agents. However, there was no scientific evaluation of the anticancer effect of both plant combinations. ObjectiveThis study investigated the potential anticancer effects of combined E. scaber and P. macrocarpa leaves extract on human breast cancer cells lines. Materials and methodsT47D cells were treated with the combination of E. scaber and each part of P. macrocarpa (leaves/EL, mesocarp/EM, seed/ES and pericarp/EP). T47D cells were then exposed to three ratios (1:1, 2:1, and 1:2) of the best combination for 24, 48, and 72 h. The cell viability of T47D and TIG-1 cells was assessed using WST-1 assay. The apoptotic hallmarks were determined using FITC Annexin V-PI staining and DNA fragmentation assay. The cell proliferation and cell cycle profiles were analyzed using CFSE (carboxyfluorescein succinimidyl ester) and Propidium iodide-flowcytometry assays. The relative number of p-ERα, p-Nrf2, p-PI3K, p-AKT, and p-mTOR were assessed using flow cytometry. The molecular docking analysis was also performed to confirm the mechanism of the extract in silico. ResultsThe combination of E. scaber and P. macrocarpa leaves (EL) possessed strong cytotoxic activity (p < 0.05) than other combination groups and cisplatin. EL showed selective killing only to T47D cells. EL at a ratio of 2:1 potentially suppressed the cell viability and cell division, induced apoptosis, and arrested the cell cycle of T47D cells by triple inhibiting the p-Nrf2, p-ERα, and p-PI3K/AKT/mTOR signaling pathway. Molecular docking analysis confirmed that the possible mechanism of EL to reduce T47D cell growth was by inhibiting ERα and Nrf2-complex, resulting in the reduction in the crosstalk effect of Nrf2, ERα and PI3K/AKT/mTOR pathways. ConclusionThe combination of leaf extracts from E. scaber and P. macrocarpa caused cell death in breast cancer cells T47D and not in normal cells TIG-1; hence has the potential to show anticancer efficacy in preclinical and clinical trials.

Abstract 095: Obstructive Sleep Apnea Induced Gut Dysbiosis Stimulates A Proinflammatory Response In Gut And Brain That Contribute To Hypertension

Objective: Obstructive sleep apnea (OSA) is an independent risk factor for the development of hypertension (HT). OSA induces gut dysbiosis, and a dysbiotic microbiota is causal in the development of HT. The mechanisms linking gut dysbiosis to blood pressure (BP) regulation remain unclear. Gut dysbiosis can induce a pro-inflammatory response of the host resulting in peripheral- and neuro-inflammation, key factors in the development of HT. We hypothesized that OSA induced gut dysbiosis elicits a pro-inflammatory response that promotes neuroinflammation and HT. Methods: OSA was induced in 8-week-old male rats (60 apneas/hr for 8 hrs during sleep) by inflating a tracheal balloon. Sham rats underwent balloon implantation without inflations. After 2 weeks of apneas, lymphocytes were isolated from brain, cecum, ileum, mesenteric lymph node (MLN), and spleen for flow cytometry. The role of IL-17 was tested by treating rats with a monoclonal antibody to IL-17 to neutralize circulating IL-17. To track the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches of the small intestine were labelled by injection of carboxyfluorescein succinimidyl ester (CFSE) dye. Results: Following 2 weeks of OSA we found a significant decrease in anti-inflammatory regulatory T (Treg) cells along with an increase in T H 17 (IL-17+) in the brain, cecum, and ileum (n=7, p&lt;0.05 for each). To examine the role of T H 17, we injected an IL-17 neutralizing antibody or control IgG during the 2 weeks of OSA. Compared to OSA rats receiving IgG control, neutralization of IL-17 significantly reduced BP of OSA rats (n=6, p&lt;0.05). IL-17 neutralization also resulted in a significantly increased Tregs and decreased Th1 cells in brain, cecum and ileum of OSA rats (n=6, p&lt;0.01 for each). To examine the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches were labelled with CFSE. We observed significant increases in CFSE+ Th1, Th2, and Th17 cells in the brain, MLN and spleen of OSA as compared to sham rats (n=6, p&lt;0.05). Conclusion: OSA induced gut dysbiosis is associated with a pro-inflammatory response in the gut and brain that involves IL-17 signaling. Treatment strategies to prevent gut dysbiosis may prove useful in reducing neuroinflammation and HT.

Abstract 2613: A modified Luria-Delbrück assay allows quantification of colorectal cancer persister cells’ mutation rate

Abstract When cancer cells are exposed to lethal doses of targeted therapies, the emergence of a subpopulation of drug-tolerant persister cells (DTPs) is often observed. We previously reported that colorectal cancer (CRC) cells exposed to targeted therapies activate an adaptive mutability stress response, involving DNA damage induction and a switch to low-fidelity DNA replication. Therefore, targeted treatment might lead to increased mutation rate in DTPs, but mutation rates of cancer cells during treatment have not been quantitatively assessed. Here, we combined biological experiments and mathematical modelling to characterize emergence and dynamics of DTPs. From this, we extrapolated parameters governing dynamics of cancer cells populations and designed a modified Luria-Delbrück assay on mammalian cells (MC-LD) to quantify mutations rates of CRC cells under standard growth conditions and during exposure to targeted therapy. We selected mismatch repair proficient CRC cell lines sensitive to different clinically used therapeutic agents, and derived clones to be used for experiments. By monitoring cell dynamics in drug-response growth assays, we found that CRC cells exposed to targeted therapy display a biphasic killing curve reaching a stable plateau, a pattern indicative of emergence of DTPs. By fitting model estimation to population growth assays, we predicted that, even if a subgroup of DTPs predated treatment, the majority of them emerged only upon exposure to targeted therapies. We also observed that DTPs slowly replicate under treatment, as shown by Carboxy fluorescein succinimidyl ester (CFSE) analysis and staining with 5-ethynyl-2’-deoxyuridine (EdU). We used these population dynamics parameters to design the MC-LD assay. CRC clones were plated in several 96-multiwell plates each, and after an expansion phase in standard culture conditions, treatment was added. After 3-4 weeks, a minority of wells showed growth of resistant colonies: based on the measured growth rates, we could predict that the resistant cells arose before treatment by spontaneous mutation. The remaining wells contained a homogenous population of DTPs. After several weeks of treatment, when pre-treatment resistant clones would have already emerged, late-emerging resistant colonies appeared in a subset of wells in which DTPs had previously been detected. Using the number of residual DTPs and resistant colonies to infer mutation rates, we found a 7- to 50-fold increase (depending on the cell line) in DTPs’ mutation rate compared to sensitive cells.In conclusion, we developed a new assay which allows quantitative comparisons of spontaneous and drug-induced mutation rates in cancer cells and showed that adaptive mutability in DTPs leads to increased mutation rates. This approach could be used to measure whether and how a wide range of environmental conditions affect DTP phenotype and mutation rates in mammalian cells. Citation Format: Alberto Sogari, Mariangela Russo, Simone Pompei, Mattia Corigliano, Giovanni Crisafulli, Andrea Bertotti, Marco Gherardi, Federica Di Nicolantonio, Marco Cosentino Lagomarsino, Alberto Bardelli. A modified Luria-Delbrück assay allows quantification of colorectal cancer persister cells’ mutation rate [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 2613.

Obstructive Sleep Apnea Induced Gut Dysbiosis Stimulates a Proinflammatory Response that Promotes Neuroinflammation and Hypertension Sriram Ayyaswamy, Bojun Zhang, Huanan Shi, Sharon Phillips, Robert Bryan and David Durgan Baylor College of Medicine, Department of Anesthesiology, Houston, Texas, United States

Background and HypothesisObstructive sleep apnea (OSA) is an independent risk factor for the development of hypertension (HT). We have previously demonstrated that OSA induces gut dysbiosis, and a dysbiotic microbiota is causal in the development of HT. However, the mechanisms linking gut dysbiosis to blood pressure (BP) regulation are unclear. Recent studies have demonstrated that gut dysbiosis can induce a pro‐inflammatory response of the host resulting in peripheral‐ and neuro‐inflammation, key factors in the development of HT. We therefore hypothesized that OSA induced gut dysbiosis elicits a pro‐inflammatory response that promotes neuroinflammation and hypertension.MethodsOSA was induced in 8‐week‐old male rats (60 apneas/hour for 8 hours during the sleep phase) by repeatedly inflating a surgically implanted tracheal balloon. Sham rats underwent balloon implantation without inflations. After two weeks of apneas, lymphocytes were isolated from aorta, brain, cecum, ileum, mesenteric lymph node, and spleen for assessment by flow cytometry. In studies examining the role of IL‐17, a monoclonal antibody to IL‐17 was injected every 48 hours during the 2 weeks of apneas to neutralize circulating IL‐17. To track the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches of the small intestine were labelled by microinjection of carboxyfluorescein succinimidyl ester (CFSE) dye.Summary of resultsFollowing 2 weeks of OSA we found a significant decrease in anti‐inflammatory regulatory T (Treg) cells along with an increase in TH17 (IL‐17+) in the brain, cecum, and ileum and TH1 (IFN‐γ+) in cecum and ileum (n=7, p&lt;0.05 for each). A significant increase in the Th17/Treg ratio, a sign of inflammation associated with hypertension, was observed in brain, cecum and ileum of OSA, versus sham, rats (n=7, p&lt;0.05 for each). To examine the role of TH17 cells, we injected an IL‐17 neutralizing antibody or control IgG antibody during the 2 weeks of OSA. Compared to OSA rats receiving IgG control, neutralization of IL‐17 significantly reduced BP of OSA rats after 1 and 2 weeks (n=6, p&lt;0.05 for each). Additionally, neutralization of IL‐17 resulted in a significant increase of Tregs and decreased Th1 cells and TNFα expressing cells in brain, cecum and ileum of OSA rats (n=6, p&lt;0.01 for each). To examine the distribution of lymphocytes originating from the gut, cells in the Peyer’s patches of sham and OSA rats were labelled with CFSE. We observed significant increases in CFSE+ Th1, Th2, and Th17 cells in the brain, MLN and spleen of OSA as compared to sham rats (n=6, p&lt;0.05).ConclusionOverall, we found that OSA induced gut dysbiosis is associated with a pro‐inflammatory response in the gut and brain that involves IL‐17 signaling. Our findings suggest that gut dysbiosis may serve as the trigger for widespread inflammation, and treatment strategies to prevent or reverse gut dysbiosis may prove useful in reducing neuroinflammation and HT.

Immunophenotypic and functional analysis of lymphocyte subsets in common variable immunodeficiency patients without monogenic defects.

Common variable immunodeficiency (CVID) is accompanied by various lymphocyte abnormalities believed to be mostly responsible for disease features in patients with no diagnosed monogenic defects. Here, we evaluated the association of B and T lymphocyte abnormalities with the incidence of CVID. Twenty-six genetically unsolved CVID patients were examined for B and T lymphocyte subsets by flow cytometry and CD4+ T-cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) test. We detected a reduction in total, naive, memory B cells and plasmablasts, and also total, naive, central memory and regulatory CD4+ T cells, besides naive CD8+ T cells. There was an increase in CD21low and transitional B cells, effector memory (EM) and terminally differentiated effector memory (TEMRA ) CD4+ T-cell subsets as well as total, EM, TEMRA , activated and cytotoxic CD8+ T cells among non-monogenic CVID patients. CD4+ T-cell proliferation response was reduced regarding both division index and percent divided. In conclusion, regarding the similarity of lymphocyte abnormalities between patients without genetic defects and those with monogenic defects, genetic mutations are not responsible for these specific lymphocyte changes. However, the novel correlations observed between lymphocyte alterations among genetically unsolved CVID patients may serve as a guide to predict the potential of future CVID development for hypogammaglobulinemia children.

Dermal fibroblasts promote cancer cell proliferation and exhibit fibronectin overexpression in early mycosis fungoides

BackgroundMycosis fungoides (MF) is caused by proliferation of malignant T-cells in the skin and may progress to involve blood, lymph nodes, and viscera. While the skin microenvironment is essential for the initiation and progression of MF in early stages, little is known about the impact of skin stroma on the growth and survival of malignant lymphocytes. ObjectiveWe investigated the effect of dermal fibroblasts and their product, fibronectin, on the survival and proliferation of malignant MF cells. MethodsFibroblasts and malignant MF CD4 T-cells were isolated from skin of patients with early-stage MF. Fibroblast-lymphocyte co-culture experiments and lymphocyte cultures on fibronectin-coated plates were established utilizing the cells derived from lesional skin, blood, and MF cell lines. The survival and proliferation rates of lymphocytes were assessed via Annexin V and carboxyfluorescein succinimidyl ester assays respectively. Additionally, integrin and fibronectin expressions in MF skin were assessed via immunofluorescence. ResultsWe found that dermal fibroblasts increased the proliferation rates of MF cells, but not normal skin or blood CD4 T-cells. However, fibroblasts did not rescue MF cells from apoptosis in co-cultures. In MF skin, we found an overexpression of a fibronectin isoform not normally found in healthy skin. MF cells expressed fibronectin-binding integrins and adhered to fibronectin but did not exhibit adhesion-mediated survival via fibronectin-integrin interactions. ConclusionOverall, our results suggest a direct role for fibroblasts, independent of fibronectin-mediated adhesion, in promoting MF cell proliferation. These findings have implications in understanding and targeting the malignant skin stromal microenvironment in cutaneous lymphomas.