三维基质侵袭实验

Yvette  May  Coulson-Thomas; Vivien  Jane  Coulson-Thomas

doi:10.21769/BioProtoc.2195

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Peer-reviewed

3D Stroma Invasion Assay

三维基质侵袭实验

YC Yvette May Coulson-Thomas email

Vivien Jane Coulson-Thomas email

发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2195 浏览次数: 8646

评审: HongLok LungAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Cell and Tissue Research, featuring study using the protocol.

Nov 2011

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Abstract

We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson-Thomas et al., 2011) and is described here in more detail.

Keywords: 3D culture (三维培养)

Fibroblasts (成纤维细胞)

Cancer cells (癌细胞)

Desmoplastic reaction (促结缔组织增生性反应)

Cancer cell invasion (癌细胞侵袭)

Stromagenic system (基质生成系统)

Background

The progression of cancer relies on intricate cross-talk between the cancer cells and surrounding cells, such as fibroblasts, inflammatory cells and endothelial cells, which form the cancer microenvironment. Fibroblasts are the major extracellular matrix producing cells and are responsible for the structural formation of tissues. Fibroblasts surrounding tumors are ‘activated’ by cancer cells into tumor-associated fibroblasts (TAFs) and play key roles in tumorigenesis and metastasis. In some cancers, TAFs up-regulate extracellular matrix expression producing an unorganized matrix, consisting mainly of collagen fibers and proteoglycans, which affects cancer cell proliferation, migration and spread. This is called the desmoplastic reaction, and during cancer cell growth different tumors may exhibit various grades of desmoplasia.

Materials and Reagents

Nunc^TM Lab-Tek^TM II Chamber Slide^TM System with 2 wells (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 154461 )
Nunc^TM cell culture dishes (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 172931 )
Cotton swabs
Human colorectal fibroblasts CCD-112CoN (ATCC, catalog number: CRL-1541 )
Caco-2 and HCT 166 cancer cell lines isolated from primary colorectal tumors (ATCC, catalog numbers: HTB-37 and CCL-247 )
pEGFP-N1 (TaKaRa Bio, Clontech)
DMEM culture medium (Thermo Fisher Scientific, Gibco^TM)
RPMI culture medium (Thermo Fisher Scientific, Gibco^TM)
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM)
L-glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 25-030-081 )
Penicillin/streptomycin (Thermo Fisher Scientific, Invitrogen^TM)
L-ascorbic acid (Sigma-Aldrich, catalog number: A4403 )
Collagen I
Anti-fibronectin (BD transduction laboratories)
FuGENE^® HD transfection reagent (Promega, catalog number: E2311 )
Trypsin/EDTA (Thermo Fisher Scientific, Gibco^TM)
Paraformaldehyde, aqueous solution - 16% (Electron Microscopy Sciences, catalog number: 15700 )
Complete media for Caco-2 and HCT 166 cells (see Recipes)
Complete media for fibroblasts (see Recipes)
Media for maintaining 3D cultures (see Recipes)

Equipment

Ultra-fine forceps with a straight tip (Fine Science tools, catalog number: 11399-80 )
CO₂ cell culture incubator (Thermo Fisher Scientific, Thermo Scientific^TM, model: Heracell^TM 150i , catalog number: 51026280)
Table top centrifuge (Eppendorf, model: 5702 RH )
Vi-CELL XR cell counter (Beckman Coulter)
Biological safety cabinets (Thermo Fisher Scientific, Thermo Scientific^TM, model: Safe 2020 Class II , catalog number: 51026639)
Scanning confocal inverted microscope (Zeiss, model: LSM 510 )
Time-lapse confocal microscope (Zeiss, model: LSM 710 )