An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist

Ibraheem, Khalidah; Dunnill, Christopher; Ιωάννου, Μαρία; As, Mohamed; Albarbar, Balid; Georgopoulos, Nikolaos T.

doi:10.21769/bioprotoc.2907

Cited by 4 publications

(15 citation statements)

References 7 publications

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“…To investigate if signal ‘quality’ (extent of receptor cross-linking) is critical in inducing RCC cell death, we treated the panel of RCC lines with membrane-CD40L (mCD40L). We utilised a culture system 22 which involves co-culture of target (epithelial) cells with effectors (fibroblasts) expressing mCD40L, alongside non-ligand expressing fibroblasts (Control). We performed pre-titration experiments to (a) determine optimal target:effector ratios (0.6:1, 0.8:1 and 1:1) and (b) assess cell death at different time-points (24, 48, 72 h) post-receptor ligation (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…ACHN, 786-O and A-704 lines were from the ATCC, supplied via Sigma (Sigma, Dorset, UK) or LGC Standards (LGC, Middlesex, UK) and were adapted for culture in DR medium (1:1 v/v mixture of DMEM and RPMI), supplemented with 5% fetal bovine serum (FBS) (Sigma). EJ and HCT116 lines were cultured as previously 22 . 3T3Neo and 3T3CD40L fibroblasts were maintained in DR/10% FBS and DR/10% FBS supplemented with 0.5 mg/ml G418 (Invivogen, supplied by Source BioScience LifeSciences), respectively, with omission of the antibiotic during co-culture experiments (below).…”

Section: Methodsmentioning

confidence: 99%

“…Unless otherwise stated, cells were seeded at 0.8 × 10 4 cells/well in 96-well plates or 5 × 10 4 cells/well in 24-well plates, and following overnight incubation they were treated with G28-5 mAb at 10 µg/ml cross-linked with 2.5 µg/ml goat anti-mouse Ig for 48 h. For CD40 activation by mCD40L, 3T3neo (control) and mCD40L-expressing 3T3CD40L (mCD40L) fibroblasts (effector cells) were growth-arrested by treatment with 10 µg/ml of Mitomycin C (MMC; Santa Cruz, supplied by Insight Biotechnology, Middlesex, UK) for 2 h in DR/5% medium and seeded either in 96-well plates at 1 × 10 4 cells/well for apoptosis detection assays, 10 cm 2 culture dishes at 3 × 10 6 cells/dish for protein lysate preparation, or 24-well plates at 6 × 10 4 cells/well for culture-supernatant collection. Following overnight incubation, culture medium was removed and epithelial cells were added at 0.8 × 10 4 cells/well in 96-well plates, 3 × 10 6 in 10 cm 2 dishes, or 5 × 10 4 cells/well in 24-well plates, respectively 22 .…”

Section: Methodsmentioning

confidence: 99%

“…This involved use of (a) CytoTox-Glo assay (Promega, Southampton, UK) for detection of compromisation of cell membrane integrity, (b) SensoLyte Homogenous AFC Capase-3/7 assay (Anaspec, Cambridge Bioscience, Cambs, UK) for effector caspase-3/7 activation, and (c) DNA Fragmentation ELISA (Roche Diagnostics, West Sussex, UK) for detection of fragmented DNA in culture supernatants. Full experimental details on the use of these assays for the co-culture system of effector/3T3 and target/epithelial cells have been described recently elsewhere 22 .…”

Section: Methodsmentioning

confidence: 99%

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CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis

et al. 2019

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A unique feature of CD40 among the TNF receptor (TNFR) superfamily is its exquisitely contextual effects, as originally demonstrated in normal and malignant B-lymphocytes. We studied renal cell carcinoma (RCC) in comparison to normal (human renal proximal tubule) cells, as a model to better understand the role of CD40 in epithelial cells. CD40 ligation by membrane-presented CD40 ligand (mCD40L), but not soluble CD40 agonist, induced extensive apoptosis in RCC cells; by contrast, normal cells were totally refractory to mCD40L. These findings underline the importance of CD40 ‘signal-quality’ on cell fate and explain the lack of pro-apoptotic effects in RCC cells previously, while confirming the tumour specificity of CD40 in epithelial cells. mCD40L differentially regulated TRAF expression, causing sustained TRAF2/TRAF3 induction in RCC cells, yet downregulation of TRAF2 and no TRAF3 induction in normal cells, observations strikingly reminiscent of TRAF modulation in B-lymphocytes. mCD40L triggered reactive oxygen species (ROS) production, critical in apoptosis, and NADPH oxidase (Nox)-subunit p40phox phosphorylation, with Nox blockade abrogating apoptosis thus implying Nox-dependent initial ROS release. mCD40L mediated downregulation of Thioredoxin-1 (Trx-1), ASK1 phosphorylation, and JNK and p38 activation. Although both JNK/p38 were essential in apoptosis, p38 activation was JNK-dependent, which is the first report of such temporally defined JNK-p38 interplay during an apoptotic programme. CD40-killing entrained Bak/Bax induction, controlled by JNK/p38, and caspase-9-dependent mitochondrial apoptosis, accompanied by pro-inflammatory cytokine secretion, the repertoire of which also depended on CD40 signal quality. Previous reports suggested that, despite the ability of soluble CD40 agonist to reduce RCC tumour size in vivo via immunocyte activation, RCC could be targeted more effectively by combining CD40-mediated immune activation with direct tumour CD40 signalling. Since mCD40L represents a potent tumour cell-specific killing signal, our work not only offers insights into CD40’s biology in normal and malignant epithelial cells, but also provides an avenue for a ‘double-hit’ approach for inflammatory, tumour cell-specific CD40-based therapy.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis

et al. 2019

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“…Following treatment, the drug-containing medium was removed and wells were rinsed once with PBS solution, before harvesting using 200 μl 0.25% trypsin/EDTA diluted in PBS for HaCaT, HaCaTa and NHEK and 1x TrypLE express enzyme for ORSK cells. Cells were counted, washed in FACS buffer as detailed elsewhere [23] and resuspended in 200 μl of ice-cold PBS for flow cytometry analysis. Doxorubicin or epirubicin uptake was determined by acquisition on a Guava EasyCyte 5 flow cytometer and results were analyzed using EasyCyte software (Millipore, Watford, UK).…”

Section: Flow Cytometrymentioning

confidence: 99%

Cooling-mediated protection from chemotherapy drug-induced cytotoxicity in human keratinocytes by inhibition of cellular drug uptake

et al. 2020

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Chemotherapy-induced alopecia (CIA) represents the most distressing side-effect for cancer patients. Scalp cooling is currently the only treatment to combat CIA, yet little is known about its cytoprotective effects in human hair follicles (HF). We have previously established in vitro human keratinocyte models to study the effects of taxanes and anthracyclines routinely-used clinically and reported that cooling markedly-reduced or even completely-prevented cytotoxicity in a temperature dependent manner. Using these models (including HFderived primary keratinocytes), we now demonstrate that cooling markedly attenuates cellular uptake of the anthracyclines doxorubicin and epirubicin to reduce or prevent drug-mediated human keratinocyte cytotoxicity. We show marked reduction in drug uptake and nuclear localization qualitatively by fluorescence microscopy. We have also devised a flow cytometry-based methodology that permitted semi-quantitative analysis of differences in drug uptake, which demonstrated that cooling can reduce drug uptake by up to~8-fold in comparison to normal/physiological temperature, an effect that was temperature-dependent. Our results provide evidence that attenuation of cellular drug uptake represents at least one of the mechanisms underpinning the ability of cooling to rescue human keratinocytes from chemotherapy drug-cytotoxicity, thus supporting the clinical efficacy of scalp cooling.

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A novel method for the establishment of autologous skin cell suspensions: characterisation of cellular sub-populations, epidermal stem cell content and wound response-enhancing biological properties

Peake,

Dunnill,

Ibraheem

et al. 2024

Front. Bioeng. Biotechnol.

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Introduction: Autologous cell suspension (ACS)-based therapy represents a highly promising approach for burns and chronic wounds. However, existing technologies have not achieved the desired clinical success due to several limitations. To overcome practical and cost-associated obstacles of existing ACS methods, we have established a novel methodology for rapid, enzymatic disaggregation of human skin cells and their isolation using a procedure that requires no specialist laboratory instrumentation and is performed at room temperature.Methods: Cells were isolated using enzymatic disaggregation of split-thickness human skin followed by several filtration steps for isolation of cell populations, and cell viability was determined. Individual population recovery was confirmed in appropriate culture medium types, and the presence of epidermal stem cells (EpSCs) within keratinocyte sub-populations was defined by flow cytometry via detection of CD49 and CD71. Positive mediators of wound healing secreted by ACS-derived cultures established on a collagen-based wound-bed mimic were detected by proteome arrays and quantified by ELISA, and the role of such mediators was determined by cell proliferation assays. The effect of ACS-derived conditioned-medium on myofibroblasts was investigated using an in-vitro model of myofibroblast differentiation via detection of α-SMA using immunoblotting and immunofluorescence microscopy.Results: Our methodology permitted efficient recovery of keratinocytes, fibroblasts and melanocytes, which remained viable upon long-term culture. ACS-derivatives comprised sub-populations with the CD49-high/CD71-low expression profile known to demarcate EpSCs. Via secretion of mitogenic factors and wound healing-enhancing mediators, the ACS secretome accelerated keratinocyte proliferation and markedly curtailed cytodifferentiation of myofibroblasts, the latter being key mediators of fibrosis and scarring.Discussion: The systematic characterisation of the cell types within our ACS isolates provided evidence for their superior cell viability and the presence of EpSCs that are critical drivers of wound healing. We defined the biological properties of ACS-derived keratinocytes, which include ability to secrete positive mediators of wound healing as well as suppression of myofibroblast cytodifferentiation. Thus, our study provides several lines of evidence that the established ACS isolates comprise highly-viable cell populations which can physically support wound healing and possess biological properties that have the potential to enhance not only the speed but also the quality of wound healing.

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An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist

Cited by 4 publications

References 7 publications

CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis

CD40 induces renal cell carcinoma-specific differential regulation of TRAF proteins, ASK1 activation and JNK/p38-mediated, ROS-dependent mitochondrial apoptosis

Cooling-mediated protection from chemotherapy drug-induced cytotoxicity in human keratinocytes by inhibition of cellular drug uptake

A novel method for the establishment of autologous skin cell suspensions: characterisation of cellular sub-populations, epidermal stem cell content and wound response-enhancing biological properties

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