细胞膜呈递CD40配体与可溶性激动剂激活CD40的体外共培养系统

Khalidah   Ibraheem; Christopher J. Dunnill; Myria  Ioannou; Albashir   Mohamed; Balid   Albarbar; Nikolaos T. Georgopoulos

doi:10.21769/BioProtoc.2907

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Peer-reviewed

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

细胞膜呈递CD40配体与可溶性激动剂激活CD40的体外共培养系统

KI Khalidah Ibraheem

CD Christopher J. Dunnill

MI Myria Ioannou

AM Albashir Mohamed

BA Balid Albarbar

Nikolaos T. Georgopoulos email

发布: 2018年07月05日第8卷第13期 DOI: 10.21769/BioProtoc.2907 浏览次数: 8311

评审: HongLok LungTomas AparicioPornima Phatak

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Cover of Oncogene, featuring study using the protocol.

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Abstract

One fundamental property of the TNR receptor (TNFR) family relates to how ‘signal quality’ (the extent of receptor ligation or cross-linking) influences the outcome of receptor ligation, for instance the induction of death in tumour cells. It is unequivocal that membrane-presented ligand (delivered to target cells via cell-surface presentation by co-culture with ligand-expressing third-party cells) induces a greater extent of carcinoma cell death in vitro in comparison to non-cross-linked agonists (agonistic antibodies and/or recombinant ligands). The CD40 receptor epitomises this fundamental property of TNF receptor-ligand interactions, as the extent of CD40 cross-linking dictates cell fate. Membrane-presented CD40 ligand (mCD40L), but not soluble agonists (e.g., agonistic anti-CD40 antibody), induces high level of pro-inflammatory cytokine secretion and causes extensive cell death (apoptosis) in malignant (but not normal) epithelial cells. In this article, we describe a co-culture system for the activation of CD40 by mCD40L and subsequent detection of various features of apoptosis (including cell membrane permeabilisation, DNA fragmentation, caspase activation) as well as detection of intracellular mediators of cell death (including adaptor proteins, pro-apoptotic kinases and reactive oxygen species, ROS).

Keywords: TNF receptors (TNFRs) (TNF受体 (TNFRs))

CD40 (CD40)

Receptor ligation (受体连接)

Membrane-presented ligand (细胞膜呈递的配体)

Soluble agonist (可溶性激动剂)

Caspase activation (半胱天冬酶激活)

DNA fragmentation (DNA片段化)

Immunoblotting (免疫印迹)

Background

The role of the TNFRs and their ligands in regulating cell proliferation or death in lymphoid tissues as well as in epithelial (and particularly carcinoma) cells has been under extensive research, as their ability to induce cell death (mainly via apoptosis) represents a promising target for cancer therapy. Importantly, however, there is a clear difference in the ability of TNFR agonists to trigger cell death when presented in soluble versus membrane-bound form. Soluble agonists often demonstrate relatively low cytotoxic potency when administrated as a sole treatment, whereas membrane-presented ligands appear to be superior (Albarbar et al., 2015).

In this context, CD40 represents the most prominent TNFR family member. The receptor is expressed on a variety of epithelial cells and the effect of CD40 activation is exquisitely contextual (Young and Eliopoulos, 2004). Most importantly, the ability of CD40 to induce cytostasis or cell death (apoptosis) is highly dependent on the ‘quality’ of receptor engagement (degree of receptor cross-linking). Soluble CD40 agonists (recombinant soluble CD40L or agonistic antibody) are only cytostatic or weakly pro-apoptotic and only rendered pro-apoptotic by pharmacological intervention (Bugajska et al., 2002). By contrast, membrane-presented CD40L (mCD40L) is highly pro-apoptotic and induces extensive apoptosis in carcinoma cells, when presented to target carcinoma cells on the surface of third-party cells (Georgopoulos et al., 2006 and 2007) or by mCD40L-expressing, naturally-activated immunocytes (Hill et al., 2008).

The ability of mCD40L (but not soluble agonists) to efficiently kill malignant cells, and in a tumour cell-specific fashion, reflects the two most remarkable properties of the CD40-mCD40L dyad and our recent studies have deciphered these two fundamental properties of CD40. We utilised a co-culture system that involved culture of target, carcinoma (or normal) cells with growth-arrested, third-party, effector cells engineered to express the CD40L on their surface, in order to achieve presentation of mCD40L. This allowed us to study the ability of mCD40L to induce a number of different morphological and biochemical features of apoptosis, as well as define the intracellular mediators of cell death (Dunnill et al., 2017). Here, we provide a detailed protocol for the preparation of the co-culture system for mCD40L delivery to epithelial target cells (in comparison to soluble agonist, i.e., agonistic anti-CD40 antibody) and methodologies to assess mCD40L-induced apoptosis and detection of its intracellular mediators.

Materials and Reagents

Materials
1. T75 tissue culture flasks with vent (SARSTEDT, catalog number: 83.1813.002 )
2. T25 tissue culture flasks with vent (SARSTEDT, catalog number: 83.1810.002 )
3. 96 well Nunc, white, flat bottom tissue culture multi-well plates (Thermo Fisher Scientific, catalog number: 136101 )
4. 96 well, flat bottom, Costar transparent tissue culture plates (Corning, catalog number: 3595 )
5. 96 well ELISA microplates (Greiner Bio One International, catalog number: 655101 )
6. 24 well plates (Corning, catalog number: 3526)
7. 6 well plates (Corning, catalog number: 3516)
8. Tissue culture dishes, 10 cm diameter, Nunclon with lid (Thermo Fisher Scientific, catalog number: 150350 )
9. Bijou tubes 5 ml sterile (x2,000) (SARSTEDT, catalog number: 60.9921.532 )
10. Cryopure tubes, 2.0 ml white cap (SARSTEDT, catalog number: 72.380 )
11. 5 ml serological pipettes (SARSTEDT, catalog number: 86.1253.001 )
12. 10 ml serological pipette (SARSTEDT, catalog number: 86.1254.001 )
13. 25 ml serological pipettes (SARSTEDT, catalog number: 86.1685.001 )
14. 120 ml sterile container graduated (x250) (SARSTEDT, catalog number: 75.9922.420 )
15. 250 ml Sterilin^TM containers (Thermo Fisher Scientific, catalog number: 190A )
16. 30 ml Sterilin^TM universals (Thermo Fisher Scientific, catalog number: 128AFS )
17. 2 ml aspiration pipette individually wrapped sterile Non Pyrogenic (SARSTEDT, catalog number: 86.1252.011 )
18. 5.0 ml TipOne^® Repeat Dispenser Tip (Sterile) (STARLAB, catalog number: S4761-0500 )
19. 1.25 ml TipOne Repeat Dispenser Tip (Sterile), Ind. Wrapped (STARLAB, catalog number: S4786-0125 )
20. Cell scrapers (Fisher Scientific, catalog number: FB55199 )
21. 20 ml disposable sterile syringe (BD, catalog number: 300296 )
22. 10 ml disposable sterile syringe (BD, catalog number: 302188 )
23. 1.5 ml tubes (SARSTEDT, catalog number: 72.690.001 )
24. 0.5 ml tubes (5,000) (SARSTEDT, catalog number: 72.699 )
25. 0.2 µm syringe filter sterile (SARSTEDT, catalog number: 83.1826.102 )
26. Syringe filter 0.4 μm (x50) (GE Healthcare, Whatman, catalog number: 6896-2504 )
27. Haemocytometer (Fisher Scientific, catalog number: MNK-420-010N )
28. Haemocytometer cover slips (Fisher Scientific, catalog number: MNK-504-030M )
29. X2500 Countess Chamber Slides (2,500) (Thermo Fisher Scientific, catalog number: C10314 )
30. Blue loose pipette tips (1,000 µl) (SARTEDT, catalog number: 70.762 )
31. 200 µl yellow pipette tips (SARTEDT, catalog number: 70.760.002 )
32. Neutral pipette tips (10 µl) (SARTEDT, catalog number: 70.1130 )
Cells
1. Human bladder carcinoma-derived EJ cells and colorectal carcinoma-derived HCT116 cells (obtained from the ATCC)
  Note: They were cultured in a 1:1 (v/v) mixture of DMEM and RPMI 1640 containing 5% FBS, referred to as ‘DR/5%’ medium.
2. Normal human urothelial (NHU) cells
  Note: They were established and cultured in complete KSFM as described (Bugajska et al., 2002).
3. 3T3neo and 3T3CD40L fibroblasts – stably transfected NIH3T3 derivatives generated as previously described (Bugajska et al., 2002)
  Note: They were cultured in DMEM supplemented with 10% FBS and containing 0.5 mg/ml G418, with omission of antibiotic in co-culture experiments (Georgopoulos et al., 2006).
Reagents
1. Purified water (double distilled ddH₂O)
2. D-PBS, 10x, no calcium, no magnesium (Thermo Fisher Scientific, catalog number: 14200067 )
3. Mikrozid AF liquid, 10 L canister (LAVABIS, catalog number: SF000301 )
4. G418, 100 mg/ml solution (InvivoGen, supplied by Source BioScience LifeSciences, catalog number: ant-gn-1 )
5. Keratinocyte Serum Free Medium (KSFM) and supplements (Thermo Fisher Scientific, Gibco^TM, catalog number: 17005075 )
6. RPMI-1640 Medium, with sodium bicarbonate, without L-glutamine (Sigma-Aldrich, catalog number: R0883-6X500ML )
7. Dulbecco’s Modified Eagle’s Medium (DMEM) (high glucose) with sodium bicarbonate, without L-glutamine (Sigma-Aldrich, catalog number: D6546-6X500ML )
8. Hanks' Balanced Salt Solution (HBSS) (Sigma-Aldrich, catalog number: H9394-6X500ML )
9. L-Glutamine solution (Sigma-Aldrich, catalog number: G7513-100ML )
10. Fetal bovine serum (FBS) (qualified fetal bovine serum, 500 ml) (Sigma-Aldrich, catalog number: F7524-500ML )
11. Ethylenediaminetetraacetic acid (EDTA) (Santa Cruz Biotechnology, catalog number: sc-29092 )
12. Trypsin-EDTA (Sigma-Aldrich, catalog number: T4174 )
13. Dimethyl Sulphoxide (DMSO) (Sigma-Aldrich, catalog number: D2650-100ML )
14. Mr. Frosty^TM Freezing Container (Thermo Fisher Scientific, catalog number: 5100-0001 )
15. Isopropanol (Fisher Scientific, catalog number: A415-4 )
16. MycoProbe^TM Mycoplasma detection assay (R&D Systems, catalog number: CUL001B )
17. Mitomycin C (10 mg) (Santa Cruz Biotechnology, catalog number: sc-3514B )
18. CK18 (cytokeratin 18) monoclonal antibody clone CY-90 (Sigma-Aldrich, catalog number: C8541-.2ML )
19. Goat anti Rabbit IgG IRDYE800 secondary antibody (tebu-bio, catalog number: 039611-132-122 )
20. Goat anti-Mouse IgG Alexa Fluor^® 680 secondary antibody (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-21057 )
21. TRAF-3 antibody (Santa Cruz Biotechnology, catalog number: sc-949 )
22. Phospho-ASK1 (Thr845) antibody (Cell Signalling Technology, catalog number: 3765 )
23. Agonistic anti-CD40 mAb G28-5 (used at 10 µg/ml), purified from culture supernatants of the HB-9110 hybridoma line (purchased from the ATCC)
24. Affinity-purified human serum protein-adsorbed goat anti-mouse IgG (X-linker) (used at 5 µg/ml) (Sigma-Aldrich, catalog number: M8645 )
25. Staurosporine from Streptomyces sp. (Sigma-Aldrich, catalog number: S4400-.1MG )
26. Docetaxel (Sigma-Aldrich, catalog number: 01885-5MG-F )
27. CellTiter 96^® AQ_ueous One Solution Assay (5,000 assays) (Promega, catalog number: G3581 )
28. Cellular DNA Fragmentation ELISA kit (for up to 500 tests) (Roche Diagnostics, catalog number: 11585045001 )
29. Cytotox-Glo^TM cytotoxicity assay (5 x 10 ml) (Promega, catalog number: G9291 )
30. Sensolyte^® Homogeneous AFC Caspase-3/7 Assay kit (Cambridge Bioscience, catalog number: ANA71114 )
31. CM-H2DCFDA (chloromethyl derivative of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) (Thermo Fisher Scientific, catalog number: C6827 )
32. Glycerol (Fisher Scientific, catalog number: 10795711 )
33. Sodium dodecyl sulphate (SDS) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: NP0002 )
34. Tris-HCl, powder, 1 KG (Melford, catalog number: T1513 )
35. Sodium fluoride (Acros Organics, catalog number: 424325000 )
36. Sodium pyrophosphate tetrabasic (Sigma-Aldrich, catalog number: P8010-500G )
37. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508-10G )
38. Protease inhibitor (PI) cocktail (Merck, catalog number: 535140-1 )
39. PI cocktail (New England Biolabs, catalog number: 5872S )
40. AQUAGUARD-1 solution for disinfection of water baths and CO₂ incubators (Biological Industries, catalog number: 01-867-1B ) (see Recipes)
41. 1x PBS (see Recipes)
42. PBS/EDTA solution (see Recipes)
43. Freezing medium (see Recipes)
44. FACS buffer (see Recipes)
45. 70% ethanol (see Recipes)–Ethanol, Absolute (Fisher Scientific, catalogue number: E/0650DF/P17 )
46. DR medium (see Recipes)
47. SSB buffer (see Recipes)
48. Lysis buffer (see Recipes)

Equipment

Gilson Pipettes
P1000 (200-1,000 μl)
P200 (50-200 μl)
P20 (2-20 μl)
P10 (1-10 μl)
Gilson REPETMAN electronic pipette 0.1-50 ml (Gilson, catalog number: F164503 )
Water bath (Memmert)
Boiling water bath (Grant)
-80 °C freezer
Sparkfree Refrigerator and Freezer (Labcold)
Electrophoresis Power Supply
Refrigerated centrifuge (PRISM R)
Universal 320 benchtop centrifuge (Hettich Zentrifugen)
Vortex mixer
Ultrasonic Homogenizer Sonicator
NuAire CellGard ES Biological Safety Cabinet (TripleRed)
Iso Class 5 Nuaire Autoflow IR direct heat CO₂ incubator with a HEPA filtration system at 37 °C and 5% CO₂ (TripleRed)
Odyssey^TM Infra-red Imaging system (Li-Cor)
Guava EasyCyte^TM Flow Cytometer (Millipore)
Countess II Automated Cell Counter (Thermo Fisher Scientific, catalog number: AMQAX1000 )
EVOS^TM XL Core Imaging System (Fisher Scientific)
FLUOstar OPTIMA (BMG Labtech)

Software

MARS software (BMG Labtech), Version 2.0
Guava EasyCyte flow cytometry software (Millipore), Guavasoft Version 2.6
Image Studio Life, Version 4.0
Adobe Photoshop CS, Version 8.0

Procedure

English

中文翻译

文章信息

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如何引用

Ibraheem, K., Dunnill, C. J., Ioannou, M., Mohamed, A., Albarbar, B. and Georgopoulos, N. T. (2018). An in vitro Co-culture System for the Activation of CD40 by Membrane-presented CD40 Ligand versus Soluble Agonist. Bio-protocol 8(13): e2907. DOI: 10.21769/BioProtoc.2907.