Magnetic-assisted Cell Sorting Research Articles

Abstract C055: Activation of oncogenes within Tff2 expressing cells of the pancreatic ductal glands results in increased lineage stemness preceding tumorigenesis: Insights from an inducible mouse model and derived organoids

Abstract Introduction: Adult stem cell dysregulation can initiate cancer. Pancreatic ductal glands (PDGs) act as a stem cell niche for the pancreatic main duct epithelium (MD). Previous studies have shown that Tff2+ cells within the PDGs give rise to progeny that migrate to the MD epithelium during inflammation. In human IPMN, the PDGs have been shown to comprise the basal proliferative compartment of these neoplasms. Here we investigate the effect of oncogenic activation and tumor suppressor deletion within Tff2+ PDG cells. Methods: We utilized Tff2CreERT2;Rosa26mT/mG;KrasLSL-G12D;P53fl/fl mice (TTKP) to activate oncogenes in Tff2+ cells and GFP tag this lineage in vivo and ex vivo. Cre activity was induced by tamoxifen injection in TTKP experimental or Tff2CreERT2;Rosa26mT/mG (TT) control mice. Pancreata were collected at pre-determined time points post-tamoxifen with GFP tagged, Tff2 lineage cells quantified and compared between groups at each timepoint. For ex vivo experiments, ductal cells were isolated from tamoxifen-naïve TTKP pancreata via magnetic-assisted cell sorting, grown in 3D culture and induced via 4OHT. GFP+ cells were sorted, cultured, and orthotopically implanted in athymic nude mice. Tumors were collected before reaching 20mm in diameter. Spatial RNA sequencing (spRNAseq) was performed on frozen tissue sections and analyzed with R package Seurat. Cross-species analysis utilized human PDAC scRNAseq reference datasets. Results: TTKP mice developed lineage-traced PDAC eight weeks after tamoxifen induction. In vivo lineage tracing showed a significant increase of GFP+ Tff2-lineage cells in PDGs and MD between TTKP and TT mice at all analyzed time points (4-, 6- and 8-weeks post tamoxifen, ≥ 3 mice each). GFP-positive PDGs exhibited progressive PanIN-like changes before PDAC formation. Tff2-PDG cells in TT mice function as transient amplifying cells, expanding within PDGs and to the MD during inflammation and contracting upon resolution. Oncogenic activation (sans inflammation) in Tff2-PDG cells led to sustained GFP+ cell presence in PDGs and MD, indicating increased stem-like behavior preceding neoplasia. Orthotopic organoid experiments resulted in GFP+ tumor formation. SpRNAseq analysis indicated enrichment of "Ductal cell type 2" in TTKP tumors, particularly overlying the main duct epithelium. Conclusion: Our study demonstrates that dysregulation of the Tff2-lineage in PDGs can initiate PDAC. Oncogenic activation in Tff2+ cells increases their stemness as indicated by lineage expansion in PDGs and MD preceding PanIN and PDAC formation. Orthotopic organoid implantation experiments further validate the tumor-initiating capacity of Tff2+ ductal cells. Spatial transcriptomics analysis confirms the presence of similar cell types identified in human scRNAseq studies within TTKP tumors. These findings strongly support the role of PDGs as a potential niche of origin for human PDAC and provide additional insights into an under-studied disease initiating process. Citation Format: Kyle L. McAndrews, Pinaki Mondal, Dulce Maroni, Dongdong Wang, Michael A. Hollingsworth, Sarah P. Thayer. Activation of oncogenes within Tff2 expressing cells of the pancreatic ductal glands results in increased lineage stemness preceding tumorigenesis: Insights from an inducible mouse model and derived organoids [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C055.

Analysis of the impact of handling and culture on the expansion and functionality of NK cells.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a key role in the elimination of tumor and virus-infected cells. Unlike T cells, NK cell activation is governed by their direct interaction with target cells via the inhibitory and activating receptors present on their cytoplasmic membrane. The simplicity of this activation mechanism has allowed the development of immunotherapies based on the transduction of NK cells with CAR (chimeric antigen receptor) constructs for the treatment of cancer. Despite the advantages of CAR-NK therapy over CAR-T, including their inability to cause graft-versus-host disease in allogenic therapies, a deeper understanding of the impact of their handling is needed in order to increase their functionality and applicability. With that in mind, the present work critically examines the steps required for NK cell isolation, expansion and storage, and analyze the response of the NK cells to these manipulations. The results show that magnetic-assisted cell sorting, traditionally used for NK isolation, increases the CD16+ population of NK cultures only if the protocol includes both, antibody incubation and passage through the isolation column. Furthermore, based on the importance of surface potential on cellular responses, the influence of surfaces with different net surface charge on NK cells has been evaluated, showing that NK cells displayed higher proliferation rates on charged surfaces than on non-charged ones. The present work highlights the relevance of NK cells manipulation for improving the applicability and effectiveness of NK cell-based therapies.

Abstract 15616: Single Cell Transcriptomic Profiling Identifies Ferroptosis and Oxidative Stress as Potential Drivers of Muscle Dysfunction in Pad

Introduction: Peripheral artery disease (PAD) affects >8 million people nationwide and is disabling. PAD-associated myopathy causes weakness yet is understudied. Unbiased transcriptomic profiling of muscle satellite cells (MuSC) which drive recovery may elucidate the etiology of ischemic myopathic change. Hypothesis: MuSC in PAD differentially express injury associated pathways that may be novel targets for intervention to reduce ischemic myopathy. Methods: Muscle tissue biopsies were obtained during lower extremity amputation or bypass in PAD/ischemic (N=4; ABI<0.6) and perfused conditions (N=4). CD56+ magnetic assisted cell sorting (MACS)-purified MuSCs were processed for single-cell RNA sequencing using the 10X Genomics platform. Unsupervised clustering and differential gene expression were performed. Western blot (WB) informed by transcriptomics confirmed pathway activation in perfused, ischemic and PAD(-) cells. Results: Among 25,298 analyzed ischemic and perfused cells, a unique cluster of MuSC transcriptionally programmed for apoptosis was detected under chronically ischemic PAD but not control conditions. Furthermore, quiescent cells with renewal capacity were lower in MuSC from ischemic muscle (17.8% vs. 26.3%, P =0.03) while pathologic myofibroblasts were almost exclusively seen in PAD (Fig.1A-B). Genes associated with iron metabolism (FTL, FTH), ferroptosis regulation (GSS), oxidative stress (HMOX) and the damage associated molecular pattern (DAMP) HMGB1 were upregulated in ischemic MuSC shown in violin plots (Fig.1C). By WB, CISD1 protein which regulates ferroptosis was elevated in PAD (p <0.03Fig.1C). Conclusions: The data implicate oxidative damage and iron-mediated programmed cell death as potential mechanisms driving muscle dysfunction from chronic ischemia. Targeting ferroptosis and oxidative stress represent novel ways to improve outcomes by reducing myopathy in PAD.

Cell sorting microbeads as novel contrast agent for magnetic resonance imaging

The success of several cell-based therapies and prevalent use of magnetic resonance imaging (MRI) in the clinic has fueled the development of contrast agents for specific cell tracking applications. Safe and efficient labeling of non-phagocytic cell types such as T cells nonetheless remains challenging. We developed a one-stop shop approach where the T cell sorting agent also labels the cells which can subsequently be depicted using non-invasive MRI. We compared the MR signal effects of magnetic-assisted cell sorting microbeads (CD25) to the current preclinical gold standard, ferumoxytol. We investigated in vitro labeling efficiency of regulatory T cells (Tregs) with MRI and histopathologic confirmation. Thereafter, Tregs and T cells were labeled with CD25 microbeads in vitro and delivered via intravenous injection. Liver MRIs pre- and 24 h post-injection were performed to determine in vivo tracking feasibility. We show that CD25 microbeads exhibit T2 signal decay properties similar to other iron oxide contrast agents. CD25 microbeads are readily internalized by Tregs and can be detected by non-invasive MRI with dose dependent T2 signal suppression. Systemically injected labeled Tregs can be detected in the liver 24 h post-injection, contrary to T cell control. Our CD25 microbead-based labeling method is an effective tool for Treg tagging, yielding detectable MR signal change in cell phantoms and in vivo. This novel cellular tracking method will be key in tracking the fate of Tregs in inflammatory pathologies and solid organ transplantation.

Polymer-Coated Magnetic Microspheres Conjugated with Growth Factor Receptor Binding Peptides Enable Cell Sorting.

The separation and sorting of human cells is an important step in the bioprocessing of cell-based therapeutics. Heterogeneous mixtures of cells must be sorted to isolate the desired cell type and purify the final product. This process is often achieved by antibody-based sorting techniques. In this work, we demonstrate that magnetic microspheres may be functionalized with peptides that selectively bind to cells on the basis of their relative concentration of specific surface proteins. Five-micrometer-magnetic microspheres were coated with the synthetic copolymer PVG (poly(poly(ethylene glycol)methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) and functionalized with the vascular endothelial growth factor receptor binding peptide (VRBP), which binds to the vascular endothelial growth factor receptor (VEGFR). These microspheres exhibited low cytotoxicity and bind to cells depending on their relative surface protein expression. Finally, coated, magnetic microspheres were used to separate heterogeneous populations of cells dependent on their VEGFR expression through magnetic-assisted cell sorting (MACS), demonstrating that peptide-based cell sorting mechanisms may be useful in the bioprocessing of human-cell-based products.

Nanobody-based CTLA4 inhibitors for immune checkpoint blockade therapy of canine cancer patients

Cancer is the leading cause of death in the geriatric dog population. Currently, the use of immune checkpoint inhibitors (ICIs) such as anti-CTLA4 antibodies has markedly improved the prognosis of several cancers in their advanced stages. However, ICIs targeting CTLA4 blockade to treat canine cancer patients are yet to define. In this study, we sought to develop, characterize and assess whether chimeric heavy chain only antibodies (cHcAbs) against CTLA4 are viable therapeutic candidates for the treatment of canine cancers. Anti-CTLA4 nanobodies (Nbs) were identified from a yeast nanobody (Nb) library using magnetic-assisted cell sorting (MACS) and flow cytometry. cHcAbs were engineered by genetically fusing the DNA sequences coding for anti-CTLA4 Nbs with the Fc domain of the subclass B of canine IgG. Recombinant cHcAbs were purified from ExpiCHO-S cells. Stable cell lines expressing canine CTLA4 and FcγRI were used to elucidate the binding ability and specificity of cHcAbs. PBMCs isolated from healthy dogs were used to evaluate the ability of cHcAbs to activate canine PBMCs (cPBMCs). Novel Nbs were identified using the extracellular domain of canine CTLA4 protein to screen a fully synthetic yeast nanobody library. Purified Nbs bind specifically to natïve canine CTLA4. We report that chimeric HcAbs, which were engineered by fusing the anti-CTLA4 Nbs and Fc region of subclass B of canine IgG, were half the size of a conventional mAb and formed dimers. The chimeric HcAbs specifically binds both with canine CTLA4 and Fcγ receptors. As the binding of Nbs overlapped with the MYPPPY motif of canine CTLA4, these Nbs were expected to sterically disrupt the interaction of canine CTLA4 to B-7s. Like their human counterpart, canine CTLA4 was expressed on helper T cells and a small subset of cytotoxic T cells. Canine Tregs also constitutively expressed CTLA4, and stimulation with PMA/Ionomycin dramatically increased expression of CTLA4 on the cell surface. Stimulation of cPBMCs in the presence of agonistic anti-CD3 Ab and cHcAb6 significantly increased the expression of IFN-γ as compared to the isotype control. This study identifies a novel nanobody-based CTLA4 inhibitor for the treatment of canine cancer patients.

A lab-on-a-chip model of glaucoma.

AimsWe developed a glaucoma‐on‐a‐chip model to evaluate the viability of retinal ganglion cells (RGCs) against high pressure and the potential effect of neuroprotection.MethodsA three‐layered chip consisting of interconnecting microchannels and culture wells was designed and fabricated from poly‐methyl methacrylate sheets. The bottom surface of the wells was modified by air plasma and coated with different membranes to provide a suitable extracellular microenvironment. RGCs were purified from postnatal Wistar rats by magnetic assisted cell sorting up to 70% and characterized by flow cytometry and immunocytochemistry. The cultured RGCs were exposed to normal (15 mmHg) or elevated pressure (33 mmHg) for 6, 12, 24, 36, and 48 hr, with and without adding brain‐derived neurotrophic factor (BDNF) or a novel BDNF mimetic (RNYK).ResultsMultiple inlet ports allow culture media and gas into the wells under elevated hydrostatic pressure. PDL/laminin formed the best supporting membrane. RGC survival rates were 85%, 78%, 70%, 67%, and 61% under normal pressure versus 40%, 22%, 18%, 12%, and 10% under high pressure at 6, 12, 24, 36, and 48 hr, respectively. BDNF and RNYK separately reduced RGC death rates about twofold under both normal and elevated pressures.ConclusionThis model recapitulated the effects of elevated pressure over relatively short time periods and demonstrated the neuroprotective effects of BDNF and RNYK.

Column-Free Method for Isolation and Culture of C-Kit Positive Stem Cells from Atrial Explants.

Ever since the discovery of stem cells, their isolation from tissues and expansion in culture has been extensively studied due to its potential for therapeutic application. The magnetic-assisted cell sorting (MACS) method is the most widely used technique for the sorting of cells based on their cell surface markers. Though effective, the major drawbacks are high cost and the requirement for the frequent replacement of the columns. In the column-free method, the cells are sorted using the same principle of immune-magnetic isolation but does not require magnetic columns, making it cost-effective. The isolation of c-kit+ stem cells from atrial explants using column-free magnet is found to be efficient and yields homogenous population of stem cells. This method saves time and labor and is economical when working with large sample sizes.

Specific inhibition of Notch1 signaling suppresses properties of lung cancer stem cells.

Lung cancer is the leading cause of cancer-related death worldwide with a relatively low 5-year relative survival rate of 16%. Novel and efficient therapeutic approach for lung cancer is desperately needed. Targeting cancer stem cells (CSCs) provides a promising strategy to eradicate malignancies. The Notch signaling pathway plays an important role in the control of cell fates and developmental processes including CSCs. The function of Notch1 in the regulation of CSCs and whether targeting Notch1 could be a potential therapy for lung cancer were explored in this study. Lung CSCs (LCSCs) were isolated from A549 cells and identified as CD44+/CD24- cells by magnetic-assisted cell sorting, then the putative LCSCs were treated with Notch1 inhibitor and Notch1 small interfering RNAs (siRNAs); the growth and proliferation of LCSCs were investigated to test the effect of Notch1 blocking on the growth and viability of LCSCs. CD44+/CD24- cells isolated from A549 cells possessed stem cell-like properties with high expression of Notch1. Blocking Notch1 by inhibitor DAPT or siRNA both inhibited the growth capacity of LCSCs. Our discovery demonstrated a depression of growth in CD44+/CD24- and A549 cells caused by blockade of Notch signaling pathway. Further studies are needed to demonstrate the detailed effects of Notch1 blocking on the LCSCs. Nevertheless, targeting the Notch pathway has exhibited great potential to be an improved lung cancer treatment.

Screening of five Sri Lankan endemic plants for anti-cancer effects on breast cancer stem cells isolated from MCF-7 and MDA-MB-231 cell lines

Purpose: To screen selected endemic plants grown in Sri Lanka on breast cancer stem cells (bCSCs) for their anti-cancer propertiesMethods: Breast-CSCs expressing CD24-/CD44+ surface markers were isolated from MDA-MB-231 cells by magnetic-assisted cell sorting method and validated using flow cytometry. A panel of forty extracts from barks and leaves of Doona nervosa, Garcinia quaesita, Garcinia zeylanica, Memecylon rostratum and Schumacheria castaneifolia were obtained by sequential solvent extraction and tested on bCSC-mammospheres derived from MDA-MB-231 and MCF-7 cells and normal mammary epithelial (MCF-10A) cells. Proliferation and cell stemness were analyzed using WST-1 cell proliferation assay and alkaline phosphatase assay.Results: Hexane and chloroform extracts of G. zeylanica and G. quaesita barks showed dosedependent reduction in proliferation and stemness in both bCSCs tested with less effect on MCF-10A cells. Hexane, chloroform and ethyl acetate extracts of S. castaneifolia bark selectively inhibited mammospheres of triple negative bCSCs cells.Conclusion: This study demonstrates that the non-polar extracts of G. zeylanica and G. quaesita, S. castaneifolia barks inhibit the proliferation of bCSCs of triple negative and estrogen-progesterone positive breast cancers. Findings of the present study may useful for developing a future anti-cancer therapeutics which can target bCSCs.Keywords: Cancer stem cells, Garcinia zeylanica, Garcinia quaesita, Schumacheria castaneifolia, Mammospheres

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent giant cell arteritis

BackgroundGiant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies but symptoms and signs are variable thereby making it a challenging disease to diagnose. A temporal artery biopsy is the gold standard to confirm GCA, but there are currently no specific biochemical markers to aid diagnosis. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA.MethodsWe recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, and aimed to collect blood samples at six time points: acute phase, 2–3 weeks, 6–8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting. RNA was extracted from all 195 collected samples for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls.ResultsOver the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, two transcripts remained differentially expressed after 12 months; SGTB, associated with neuronal apoptosis, and FCGR3A, associatied with Takayasu arteritis. We detected genes that correlate with both symptoms and biochemical markers used for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes including Polymyalgia Rheumatica, bilateral blindness and death within 12 months.ConclusionsThis is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide in initiating appropriate patient management.

Influence of Carbohydrate Ingestion from Bananas or Sugar Beverage on Natural Killer Cells During Recovery from 75‐km Cycling

Natural killer cells are part of the first line of defense against infection. They are also the most responsive of all immune cell types to acute exercise bouts, and their depletion from the bloodstream and potential cytotoxic capabilities loss during the recovery phase open a window for opportunistic infections to develop. Carbohydrate intake has multiple effects for the exercising athlete, including enhanced glucose availability to the working muscle, resulting in reduced post‐exercise cortisol and epinephrine levels, decreased leukocytosis and phagocytosis, and lower inflammatory cytokines. The effect of carbohydrate intake on post‐exercise NK bioactivity has never been directly assessed, and the purpose of this study was to compare ingestion of Cavendish bananas with an equicaloric, sugar‐only beverage or water alone on NK cell release and cytotoxic activity during recovery from an intensive 75‐km cycling bout. Participants included 16 male and female cyclists (age 39.9±2.4 years) who regularly competed in road races and were capable of cycling 75‐km at race pace. This study utilized a randomized crossover approach, and participants engaged in three 75‐km cycling time trials while ingesting water only, bananas and water, and a 6% sugar beverage in an overnight fasted state, with data analyzed using a repeated measures two‐way analysis of variance (ANOVA), or paired two‐tailed student t‐test. Before the exercise bout, participants ingested 5 ml/kg water alone, 6% sugar beverage, Cavendish (with water) adjusted to quantities providing 0.4 g carbohydrate per kilogram of body weight (g carb/kg). During the cycling bout, water was ingested at a rate of 3 ml/kg every 15 minutes, with carbohydrate intake from the 6% sugar beverage and banana adjusted to 0.2 g carb/kg every 15 minutes. Blood samples were collected pre‐ and post‐exercise, and then at 1.5 h and 21 h post‐exercise. Isolation and count of NK cells from whole blood was done using a Magnetic Assisted Cell Sorting technology (Miltenyi, Germany) using a CD56 positive selection. NK cell cytotoxicity was assessed using an Amnis Imagestream flow cytometer (Millipore, MA). NK cell count profile was significantly different in the water group compared to all the other groups (group interaction p=0.0051 by ANOVA), with post‐exercise NK cell counts about 71% higher in the water‐treated group vs all other groups (p&lt;0.05 by Student t‐test). NK cell cytotoxicity was significantly decreased about 70% 1.5 h post‐exercise for all treatments, with no trial differences (time effect, p=0.0057 by ANOVA). In conclusion, these data indicate that carbohydrate ingestion from bananas with water or a 6% sugar beverage compared to water alone attenuates the sharp post‐exercise release of NK cells in the blood compartment, but has little effect on countering the post‐exercise decrement in NK cell cytotoxic potential.Support or Funding InformationSupport provided by Dole Foods, Inc., Westlake Village, CA

0177 Bovine macrophage phenotype influences inflammatory response to lipopolysaccharide

Severe inflammation during gram negative bacterial disease is common in periparturient dairy cows and increases the severity of diseases such as Escherichia coli mastitis. Tissue inflammation is partly orchestrated by macrophage responses to bacterial infection. Studies in monogastric species showed classical phenotype macrophages have proinflammatory responses and alternative phenotype macrophages have protective and restorative responses during disease. However, responses of diverse bovine macrophage phenotypes to lipopolysaccharide are unclear. The objective of this research was to compare the lipopolysaccharide-induced inflammatory response in several phenotypes of bovine primary monocyte-derived macrophages. Peripheral blood mononuclear cells were isolated from whole blood using Ficoll (n = 8 cows). Monocytes were identified using mouse anti-bovine CD172α monoclonal antibody and separated from lymphocytes using magnetic assisted cell sorting. Monocytes were cultured with interferon-γ or interleukins (IL) 4 and 13 to induce a classical or alternative macrophage phenotype, respectively, then stimulated with lipopolysaccharide. Macrophage mRNA was quantified in adipose using qPCR. Fold changes in mRNA concentration were calculated by 2-ΔΔCt, using the untreated cells as calibrator and three endogenous control mRNA. Treatment differences in mRNA expression were identified using Fisher pairwise comparisons and ANOVA (P ≤ 0.05). Flow cytometry showed magnetic assisted cell sorting increased CD172α+ cells from 22.3 ± 1.9 to 81.6 ± 2.8%. After 48 h in vitro, CD68 expression increased and CD172α+ was 95.2 ± 0.4%. Lipopolysaccharide increased IL6, IL10, TNF, and CCL2 expression. Lipopolysaccharide stimulated IL6 and IL10 expression was decreased in alternative macrophages, whereas lipopolysaccharide stimulated TNF expression was increased in classical macrophages. Lipopolysaccharide stimulated CCL2 expression was not different between macrophage types. Together these results show an exacerbated proinflammatory cytokine profile in a model of classical bovine macrophages during gram negative bacterial disease. Results suggest that macrophage phenotype could be involved with severe inflammatory responses seen during dairy cow periparturient periods characterized by prolonged and exacerbated lipolysis and increased disease susceptibility. Ongoing research will describe macrophage phenotype during bovine disease and identify factors contributing to phenotype change. Such factors could ultimately be manipulated to control the bovine macrophage inflammatory response.

Abstract P615: Hypertension-Dependent Reprogramming of the Endothelial Transcriptome

Hypertension has drastic consequences for the cardiovascular system and especially for endothelial cell health. Clinically this is observed through the repeated finding that hypertension is associated with endothelial dysfunction. Despite this strong evidence that hypertension leads to impairment of endothelial function, the molecular events that give rise to endothelial dysfunction are poorly characterized. To better understand how hypertension affects the health and function of endothelial cells at the molecular level, we utilized a mouse model of spontaneous hypertension (BPH/2J) with the overarching hypothesis that hypertension causes the transcriptional reprogramming of endothelial cells leading to endothelial dysfunction. First, we non-invasively quantified endothelial function from hypertensive and normotensive (BPN/3J) mice by measuring flow-mediated vasodilation with high-frequency ultrasound. We found that hypertensive mice failed to dilate their femoral artery while normotensive control mice dilated their femoral artery 17 ± 8% (Mean ± SEM). Next, we acutely isolated cardiac endothelial cells by magnetic-assisted cell sorting for endothelial marker CD31. Through this technique, we enriched our sample for endothelial cells (as defined by expression of two endothelial-specific cell-surface markers CD31 and CD102) to 92 ± 1% compared to 9 ± 1% in pre-magnetically sorted cells. Finally, we isolated RNA and transcriptionally profiled the endothelial cells from normotensive and hypertensive mice (n=3 mice per group) with an average depth of 30 million reads per sample with RNAseq. We found that over 4000 genes were differentially expressed between groups (FDR cutoff of .01). Using Ingenuity Pathways Analysis (Qiagen), we identified multiple pathways that were dysregulated in endothelial cells exposed to hypertension including those related to immune function, cell morphology and cell-to-cell communication. This work represents one of the first studies to utilize cell-specific RNAseq to quantify how endothelial cells are transcriptionally reprogrammed in a pathological state. It is our hope that this work will lead to targeted therapeutics to improve endothelial function in patients with hypertension.

Effect of Interferon Alfa-2a on Peripheral Blood CD4+CD25+ T Regulatory Cells in Patients with Behcet Uveitis: Preliminary Study

Purpose: To evaluate the phonotypical and functional effect of interferon alfa-2a therapy on peripheral blood CD4+CD25+ T regulatory (Treg) cells in patients with Behcet uveitis. Methods: Blood was taken from 5 patients with refractory Behcetpanuveitis and 5 age-matched healthy controls. Flow cytometric analysis of CD4+CD25+Treg cells was performed. CD4+CD25+Treg cells were separated by magnetic-assisted cell sorting and co-cultured. Cytokine levels in the supernatants were determined by ELISA. Results: The percentage of CD4+CD25+Foxp3+Treg cells and the intensity of Foxp3 expression of CD4+CD25+Treg cells were slightly elevated in patients when compared to controls. Interferon alfa-2a led to a borderline significant decline of CD4+CD25+Foxp3+Treg cells and elevation of interleukin-10 (p=0.06). Conclusions: Interferon alfa-2a therapy might lead to a decline in the dysfunctional CD4+CD25+Foxp3+Treg cell population. Interleukin-10 may play a major role in IFN α-2a mediated control of Behcet uveitis.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with foetal bovine serum (10% v/v), d-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32–80 years) or duration in eye bank corneal preservation medium prior to use (3–10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p < 0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Enrichment of spermatogonial stem cells from long-term cultured human testicular cells

To evaluate the degree of enrichment of spermatogonial stem cells (SSCs) from human testicular cell cultures by ITGA6+, HLA-/ITGA6+, GPR125+, and HLA-/GPR125+ magnetic-assisted cell sorting (MACS). Experimental basic science study. Reproductive biology laboratory. Multiple samples of cryopreserved human testicular cells from two prostate cancer patients with normal spermatogenesis. Cultured human testicular cells subjected to four sorting strategies based on MACS and xenotransplanted to the testes of mice to determine the enrichment for SSCs. Enrichment for human spermatogonia and SSCs tested by expression analysis of spermatogonial markers ITGA6, GPR125, ZBTB16, UCHL1, and ID4 using quantitative real-time polymerase chain reaction (qPCR) and by xenotransplantation into the testes of mice, respectively. Compared with the nonsorted cultured testicular cells, only the ITGA6+ and HLA-/GPR125+ sorted cells showed enrichment for ID4. No difference in expression of ZBTB16 and UCHL1 was observed. Xenotransplantation of the sorted cell fractions showed a 7.1-fold enrichment of SSCs with ITGA6+. Magnetic-assisted cell sorting of cultured human testicular cells using ITGA6 allows for enrichment of SSCs, which aids in further molecular characterization of cultured human SSCs and enhances testicular colonization upon transplantation in future clinical settings.

Epigenetic modulation of glucocorticoid receptors in posttraumatic stress disorder

Some individuals suffering from posttraumatic stress disorder (PTSD) exhibit lower basal salivary cortisol and higher glucocorticoid receptor (GR) sensitivity. Recent studies suggest that epigenetic mechanisms regulate the activity of cortisol and GR. As a means to combine and cross-validate those findings, we compared cortisol, GR expression and promoter methylation levels in peripheral T lymphocytes of healthy controls versus individuals endorsing a diagnosis of lifetime PTSD. Thirty subjects with lifetime (current or remitted) PTSD and 16 subjects never exposed to trauma were recruited. Salivary cortisol was collected at six time points over the course of a single weekday and analyzed utilizing a time-resolved fluorescence immunoassay. GR expression (GRtotal, 1B, 1C, 1F and 1H) was measured by quantitative RT-PCR. DNA methylation levels in human glucocorticoid receptor (hGR) 1B and 1C variant's promoter were quantified by epityper in T lymphocytes isolated by magnetic-assisted cell sorting. Individuals with lifetime PTSD have lower morning cortisol release, higher mRNA expression of hGRtotal, 1B, and 1C and lower overall methylation levels in hGR 1B and 1C promoters. Cortisol levels were inversely correlated with hGR 1B mRNA expression. Moreover, overall and CpG site-specific methylation levels were inversely correlated with hGRtotal and 1B mRNA expression. There was no difference between current and remitted PTSD across cortisol, GR expression mRNA and DNA methylation data. Traumatic events induce DNA methylation alterations in distinct promoters of hGR with transcriptional modifications that associate with hypoactive hypothalamus-pituitary-adrenal axis in individuals with PTSD. Our results also point toward an important role of hGR 1B variant in PTSD.

MYC Translocations In Multiple Myeloma Involve Recruitment Of Enhancer Elements Resulting In Over-Expression and Decreased Overall Survival

Myc deregulation is an important and recurrent molecular event in myeloma. MYC is located at chromosome 8q24 and gene-mapping analyses of copy number abnormalities (CNA) within the myeloma genome have identified a hot spot of activity at this locus. We have previously found evidence of CNA at 8q24 in 9% of 114 presenting cases. The presence of recurrent copy number variants at 8q24 suggest either an important underlying molecular mechanism active at this locus, that is important in the pathogenesis of myeloma, or that deregulation of MYC is an important recurrent molecular event in myeloma. In order to address the prevalence and mechanism of rearrangements at the 8q24 locus and their association with chromosomal translocations into the Ig gene loci we used a massively parallel sequencing approach combined with pull down of the region surrounding the MYC locus as well as the Ig loci to define the molecular mechanism active at this locus and their association with other better described translocations.CD138-positive bone marrow plasma cells were selected to a purity >95% using magnetic assisted cell sorting (Miltenyi Biotech). We developed a targeted capture using the SureSelect (Agilent) system by tiling RNA baits across the MYC, IGH, IGK and IGL loci. The region captured surrounding MYC spanned from 127.5-129.8 Mb on chromosome 8, which is roughly 1 Mb either side of MYC which is located at 128.75 Mb. This region includes POU5F1B and PVT1 which are common sites of translocations in myeloma. Repetitive sequences in the MYC region were masked resulting in an overall capture of 66% of the sequence. DNA from 104 samples were assayed using 150 ng of DNA and a modified capture protocol with eight cycles of pre-hybridization PCR and 11 cycles of post-hybridization PCR. Samples were barcoded using Illumina indexes and up to 27 samples were sequenced per lane on a HiSeq2000 generating 76-bp paired-end reads. After base calling and quality control metrics, the raw fastq reads were aligned to the reference human genome (build GRCh37) resulting in a median depth of 289X per sample after de-duplication for the captured region. Translocation breakpoints were identified in the sequencing data using SVDetect. Bioinformatically called breakpoints were further filtered based on depth, unique mappability, number of supporting reads and whether or not they were detected in non-tumor samples.Of the 104 samples interrogated, 8q24 breakpoints were found in 22 (21%). These samples comprised of t(11;14) (n=6), t(14;16) (n=10), t(14;20) (n=1), t(6;14) (n=1), t(7;14) (n=1), and those with no IGH translocation (n=3). There was an enrichment for t(14;16) (10/17) and t(11;14) (5/27) and a depletion of t(4;14) (0/13) and hyperdiploid (3/28) with an 8q24 breakpoint. Eight of the 22 samples had an 8q24 translocation involving IGH, IGK or IGL. The remaining samples with 8q24 breakpoints had partner genes scattered throughout the genome, but with a preponderance for those associated with B cell biology or known myeloma-associated genes. These genes included CCND1, FAM46C, XBP1, KRAS and FOXO3. In all samples the position of the breakpoints on 8q24 were spread out over 1.2 Mb. The majority were situated telomeric of PVT1 (51.5%) with 12% within PVT1, 15% between MYC and PVT1 and 15% between POU5F1B and MYC. Of the 3 samples with breakpoints within PVT1 2 have potential fusion genes, one with FOXO3 and one with LINC00309. Nine samples had two breakpoints on 8q24, often with two different chromosome partners. Several samples had complex rearrangements resulting in multiple chromosomal segments being joined together, often involving MYC, an Ig enhancer and the primary Ig partner oncogene (e.g. CCND3). Given that 8 samples have translocations involving MYC and Ig loci, which presumably results in overexpression of MYC driven by the Ig loci enhancers, we looked for enhancer sequences in the partner chromosomes of the other samples. Most samples had easily identifiable B cell H3K27Ac marks (taken from ENCODE), identifying active regulatory elements. 55 samples were taken from the MRC Myeloma IX trial and a comparison of those with and without MYC breakpoints showed a significant decrease in overall survival (P=0.04) in those with a breakpoint (median of 21.5 versus 55.8 months).We conclude that MYC translocations occur in up to 21% of presenting myeloma samples and involve the sequestration of active enhancer elements, resulting in increased expression of MYC. Disclosures:No relevant conflicts of interest to declare.

Genome-wide Profiling in AML Patients Relapsing after Allogeneic Hematopoietic Cell Transplantation

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.