化疗相关性粘膜炎与口腔机会性感染模型的建立

Takanori Sobue; Martinna Bertolini; Angela Thompson; Anna Dongari-Bagtzoglou

doi:10.21769/BioProtoc.3411

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Model of Chemotherapy-associated Mucositis and Oral Opportunistic Infections

化疗相关性粘膜炎与口腔机会性感染模型的建立

TS Takanori Sobue email

MB Martinna Bertolini

AT Angela Thompson

AD Anna Dongari-Bagtzoglou email

发布: 2019年11月05日第9卷第21期 DOI: 10.21769/BioProtoc.3411 浏览次数: 4459

评审: Kristin L. ShinglerEmmanuel Orta-ZavalzaLucy Xie

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Cover of PLOS Pathogens, featuring study using the protocol.

Apr 2019

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Abstract

Oral mucositis is a common complication of cancer chemotherapy treatment. Because of the lack of relevant oral mucositis experimental models, it is not clear how chemotherapeutic agents injure the oral mucosa and if commensal microorganisms accelerate tissue damage. We developed an organotypic oral mucosa model that mimics cellular responses commonly associated with cytotoxic chemotherapy. The organotypic model consists of multilayer oral epithelial cells growing over a collagen type I matrix, with embedded fibroblasts. Treatment of organotypic constructs with the chemotherapeutic agent, 5-fluorouracil (5-FU), leads to major histopathologic changes resembling mucositis, such as DNA synthesis inhibition, increased apoptosis and cytoplasmic vacuolation. Candida albicans formed mucosal biofilms on these tissues and augmented the inflammatory responses to 5-FU. This model can be used in further mechanistic studies of oral mucositis and associated opportunistic infections.

Keywords: Oral mucositis (口腔粘膜炎)

Background

Candida albicans is a fungal commensal of the upper and lower gastrointestinal tract implicated in opportunistic mucosal and disseminated infections in immunocompromised hosts (Westbrook et al., 2013). Clinical studies have shown that risk factors associated with candidemia go beyond the frequently described use of central venous catheters; they are also associated with the use of high dose chemotherapeutic agents. Under this scenario, the mucosal barrier integrity is decreased, resulting in life threatening systemic dissemination (Nucci et al., 1998; Lalla et al., 2014). We have recently started to explore the role of cytotoxic chemotherapy in Candida albicans mucosal infection. We showed that this fungus can amplify the proinflammatory epithelial response to the cytotoxic drug 5-FU in vitro, (Sobue et al., 2018) and it can promote a dysbiotic state in vivo, that causes increased oral mucosal barrier breach (Bertolini et al., 2019). 5-fluorouracil is a chemotherapeutic agent used for treatment of a variety of metastatic cancers including breast, gastrointestinal tract and head & neck. In this patient population oral thrush and/or candidemia with disseminated multi-organ infection are prevalent and most frequently attributed to C. albicans (Teoh and Pavelka, 2016).

Thus far organotypic models of oral mucositis have relied on cost-prohibitive commercially available constructs (Sonis et al., 2017) or primary epithelial cultures which vary in life span and other phenotypic characteristics, and are associated with ethical considerations for harvest (Tobita et al., 2010). Our in-house organotypic construct is based on an oral epithelial cell line which readily forms a multilayer epithelial structure and responds to 5-FU in a manner similar to oral mucosa in vivo (Sobue et al., 2018; Bertolini et al., 2019). This model will facilitate further mechanistic studies on oral mucositis and the complex relationships with fungal and bacterial organisms that overgrow on the oral mucosa during chemotherapy-driven immunosuppression. This model could lead to novel future interventions designed to prevent the initiation of oral mucositis in chemotherapy treated patients and better understand the effect of resident microbiota in aggravating oral mucosal lesions.

Materials and Reagents

Plates/Inserts/tubes/pipettes
1. 6-well Falcon Deep Well Plate (Corning, catalog number: 355467)
2. Transwell 3414 (24 mm diameter inserts, 3.0 μm pore size, Corning, catalog number: 3413)
3. 50 ml polypropylene tube (Fisher Scientific, catalog number: 05-539-8)
4. 25 ml polystyrene pipette (Fisher Scientific, catalog number: 13-678-11)
5. 10 ml polystyrene pipette (Fisher Scientific, catalog number: 13-678-11E)
6. 9 inch glass pasteur pipette (Fisher Scientific, catalog number: 13-678-20C)
7. Tissue embedding cassettes (Fisher Scientific, catalog number: B1000729BL)
8. Tweezers (Fisher Scientific, catalog number: 12-000-132)
9. 75 cm² Cell culture flask (Corning, catalog number: 430641U)
10. 100 mm Petri Dish (Fisher Scientific, catalog number: FB0875713)
Cells
Note: Use freshly thawed vial of cells from Liquid Nitrogen. Sub-culture when cells are 70-80% confluent. Sub-culture one week after starting culture.
1. Fibroblast: 3T3 cell line (ATCC, catalog number: CRL-1658)
2. Epithelial cells: SCC15 cell line (ATCC, catalog number: CRL-1623)
Common reagent
1. 10% normal goat serum (Jackson ImmunoResearch, catalog number: 005-000-121)
2. 100%, 95%, 70% and 50% Ethanol (EtOH) (Pharmo, catalog number: 111000200)
3. 30% H₂O₂ (Sigma-Aldrich, catalog number: H1009-100ML)
4. Phosphate buffered saline (PBS, Corning, catalog number: 21-031-CV)
5. Triton X-100 (Sigma-Aldrich, catalog number: T8787)
6. Xylene (Fisher Scientific, catalog number: X3P-1GAL)
7. 1 N Sodium Hydroxide (NaOH, Sigma-Aldrich, catalog number: S2770)
8. Dimethyl sulfoxide (DMSO, Sigma-Aldrich, catalog number: 276855)
9. 0.05 % Trypsin-EDTA (Gibco, catalog number: 25300-054)
10. 20x Citrate buffer (Thermo Fisher Scientific, catalog number: 005000, see Recipes)
11. 16% Paraformaldehyde (Electron Microscopy Science, catalog number: 15710, see Recipes)
Fibroblast Culture Media: DMEM-10% FBS-1% Pen/Strep
1. Dulbecco's Modified Eagle Medium (DMEM) liquid (high glucose) without Pyruvate (Gibco, catalog number: 11965-084)
2. Fetal bovine serum (FBS, Gibco, catalog number: 16000044)
3. Penicillin/streptomycin (Pen/Strep, Gibco, catalog number: 15140-122)
Epithelial Cell Culture Media
KSFM with Bovine Pituitary Extract (BPE) (1 tube/each bottle), 0.2 ng/ml human epithelial growth factor (hEGF) (Invitrogen, catalog number: 17005-042), 0.4 mM CaCl₂ (Sigma-Aldrich, catalog number: C-3306) and Pen/Strep (1:100).
Matrix and organotypic cell culture media components
1. Type I Collagen (Organogenesis, catalog number: 200/50)
2. Matrigel Basement Membrane Matrix (BD Biosciences, catalog number: 354234)
3. Newborn Calf Serum (HyClone, catalog number: SH 3011802)
4. DMEM (-) (Gibco, catalog number: 11965-084)
5. Ham's F12 (Gibco, catalog number: 11765-054)
6. 10x EMEM (BioWhittaker, catalog number: 12-684F)
7. 7.5% Sodium-Bicarbonate (Na-Bicarbonate, BioWhittaker, catalog number: 17-613E)
8. FBS (Gibco, catalog number: 16000044)
9. L-Glutamine (Cellgro, catalog number: 25-005CI)
10. Gentamicin, 50 mg/ml (Cellgro, catalog number: MT30-0005-CR)
11. ITES - Insulin, Transferrin, Ethanolamine and Selenium (BioWhittaker, catalog number: 17839Z)
12. Hydrocortisone (Sigma-Aldrich, catalog number: H0888, see Recipes)
13. O-phosphorylethanolamine (Sigma-Aldrich, catalog number: P0503, see Recipes)
14. Adenine (Sigma-Aldrich, catalog number: A9795, see Recipes)
15. Progesterone (Sigma-Aldrich, catalog number: P8783, see Recipes)
16. Triiodothyronine (Sigma-Aldrich, catalog number: T5516, see Recipes)
17. 5-Fluorouracil, 5-FU (Sigma-Aldrich, catalog number: F6627, see Recipes)
18. 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside, BrdU (Sigma-Aldrich, catalog number: B4252, see Recipes)
Microorganisms used and microbiological media
1. Candida albicans strain SN425 (reference strain, derived from SC5314, provided by Dr. Clarissa J. Nobile, University of California, Merced, CA)
2. YPD medium and Agar (see Recipes)
  1. Yeast Extract (Sigma-Aldrich, catalog number: Y1625)
  2. Peptone (BD, catalog number: 211677)
  3. Dextrose (J. T. Baker, catalog number: 1616-05)
  4. Agar (BD, catalog number: 214530)
Immunofluorescence stain
1. Coplin Staining Jar (Thermo Scientific, catalog number: 19-4)
2. Rat anti BrdU monoclonal antibody (Abcam, catalog number: ab6326)
3. Goat anti rat Alexa Fluor 555 conjugated secondary antibody (Thermo Fisher scientific, catalog number: A21434)
4. Goat anti rabbit anti active Caspase-3 antibody (Abcam, catalog number: Ab32042)
5. Alexa Flour 568 conjugated secondary antibody (Thermo Fisher scientific, catalog number: A11011)
6. Rabbit FITC conjugated anti Candida albicans polyclonal antibody (Meridian Life Science, catalog number: B65411F)
7. Hoechst 33258 (10 mg/ml, Thermo Fisher scientific, catalog number: H3569)
8. Fluoro-Gel (Electron Microscopy Sciences, catalog number: 17895-11)

Equipment

Fluorescence microscope (Zeiss Axio Imager M1)
Objective lens (EC Plan-Neofluar 20x/0.5)
Tissue culture incubator (Thermo Forma, catalog number: 3110)
Biological safety cabinet (Thermo Forma, catalog number: 1284)
Autoclave
Orbital Shaker (New Brunswick Scientific, catalog number: C1A88IC-C1)
Centrifuge machine (Thermo Scientific, catalog number: CL2)
Hemocytometer (Hausser Scientific, Fisher Scientific, catalog number: 0267110)

Software

AxioVision LE64 (Zeiss)

Procedure

English

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如何引用

Sobue, T., Bertolini, M., Thompson, A. and Dongari-Bagtzoglou, A. (2019). Model of Chemotherapy-associated Mucositis and Oral Opportunistic Infections. Bio-protocol 9(21): e3411. DOI: 10.21769/BioProtoc.3411.