肿瘤相关巨噬细胞和卵巢癌细胞的3D共培养系统

Lingli Long; Mingzhu Yin; Wang  Min

doi:10.21769/BioProtoc.2815

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Peer-reviewed

3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells

肿瘤相关巨噬细胞和卵巢癌细胞的3D共培养系统

LL Lingli Long

MY Mingzhu Yin

Wang Min

发布: 2018年04月20日第8卷第8期 DOI: 10.21769/BioProtoc.2815 浏览次数: 10761

评审: Jia LiLucíola Silva BarcelosAnonymous reviewer(s)

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Abstract

Ovarian cancer is fairly unique in that ovarian carcinoma cells can detach and spread directly through peritoneal cavity. It has been unclear, however, how detached cancer cells survive in the peritoneum and form spheroid structure. We have recently reported that there is a strong correlation between Tumor-associated macrophages (TAMs)-associated spheroid and clinical pathology of ovarian cancer, and that TAMs promote spheroid formation and tumor growth at early stages of transcoelomic metastasis in orthotopic mouse models. We have established an in vitro spheroid formation assay using a 3D co-culture system in which mouse GFP⁺F4/80⁺CD206⁺ TAMs isolated from spheroids of ovarian cancer-bearing donor tomato^lysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. As transcoelomic metastasis is also associated with many other cancers such as pancreatic and colon cancers, TAM-mediated spheroid formation assay would provide a useful approach to define the molecular mechanism and therapeutic targets for ovarian cancer and other transcoelomic metastasis cancers.

Keywords: Ovarian cancer (卵巢癌)

Tumor-associated macrophage (肿瘤相关巨噬细胞)

3D co-culture system (3D共培养系统)

Spheroid formation (球体形成)

Transcoelomic metastasis (种植性转移)

Background

Ovarian cancer (OC) is the second most common gynecological cancer and the leading cause of death in the United States (Jemal et al., 2009; Siegel et al., 2012). The major reason for the poor prognosis of OC is intraperitoneal and pelvic extensive implantation metastasis, which is usually unable to be removed completely by surgery. The most widely ascribed explanation for the phenomenon of peritoneal metastasis is that tumor cells become detached from the primary tumor after extension into the peritoneal surface and are transported throughout the peritoneal cavity by peritoneal fluid before seeding intraperitoneally. It has been suggested that the process of transcoelomic metastasis could be divided into several steps: 1) cell detachment, survival and resistance of anoikis; 2) evasion of immunological surveillance; 3) epithelial-mesenchymal transition; 4) spheroid formation; 5) ascites formation; and 6) peritoneal implantation (Tan et al., 2006; Peart et al., 2015; Rafehi et al., 2016). However, it remains unclear how free detached tumor cells survive in transcoelomic environment and form spheroids at initial steps of transcoelomic metastasis. Our recent study reveals that TAMs play an essential role in the survival and proliferation of free cells detached from the primary tumor in transcoelomic environment and spheroid formation at early stages of transcoelomic metastasis (Yin et al., 2016).

One critical method in this study is an in vitro spheroid formation assay using a 3D co-culture system to determine how TAMs facilitate spheroid formation. In this assay, mouse GFP⁺F4/80⁺CD206⁺ TAMs isolated from spheroids of ovarian cancer-bearing donor tomato^lysM-cre mice were mixed with ID8 cells (TAM:ID8 at a ratio of 1:10) in medium containing 2% Matrigel and seeded onto the 24-well plate precoated with Matrigel. Similarly, we use human CD14⁺ TAMs isolated from OC patients and human ovarian cancer SKOV3 cells. In this model, we detect spheroid formation at 48 h of co-culture (Figure 1).

Here, we summarize our detailed protocols for 3D spheroid formation assay.

Figure 1. TAMs and OC cells in vitro 3D co-culture system were showed by Immunofluorescence. A. TAMs and OC cells form spheroids in an in vitro 3D co-culture system. GFP⁺F4/80⁺CD206⁺ TAMs isolated from spheroids of ovarian cancer-bearing donor tomato^lysM-cre mice and ID8 cells were co-cultured in the Matrigel-precoated 24-well plate for 48 h. The spheroids were subjected to immunofluorescent staining for E-cadherin for tumor cells. Images for GFP⁺ TAMs, E-Cadherin⁺ OC cells and DAPI for all cells in the spheroids are shown. B. Human TAMs were isolated and infected with lentivirus expressing RFP. RFP-expressing TAMs were incubated with SKOV3 human ovarian cancer cells followed by 3D co-culture for 72 h. Spheroids were immunostained with keratin-14. Scale bars = 10 μm.

Materials and Reagents

Pipette tips
15 cm Petri dish
10 ml serological pipette
50 ml sterile conical tube (Corning, Falcon^®, catalog number: 352070 )
18-gauge needle
10 ml syringe
Falcon^TM cell strainers,100 μm (Corning, Falcon^®, catalog number: 352360 )
1.5 ml microcentrifuge tubes
Greiner cellstar multiwall culture plates (24 wells, Greiner Bio One International, catalog number: 662102 )
Cell lines: ID8 ovarian cancer cell line (Yin et al., 2016) was a gift from Jack Lawler and Carmelo Nucera at Beth Israel Deaconess Medical Center (Harvard Medical School, Boston, Massachusetts, USA).
Note: ID8 cells are mouse epithelial OC line derived from C57BL/6 background; Passage under 30 and less than 1 week culture before injections.
C57BL/6 mice, female, age: 8 weeks
Tomato reporter transgenic mice: ID8 OC cells were labeled by stably expressing mCherry fluorescence protein while LysMCre mice crossed to the tomato reporter mT/mG
Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10567014 )
D-glucose
Fetal bovine serum (FBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 26140079 )
Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140 )
0.25% Trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 )
Ketamine
Bovine serum albumin (BSA) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: B14 )
Collagenase (Thermo Fisher Scientific, Gibco^TM, catalog number: 17104019 )
F4/80 monoclonal antibody, APC conjugate for flow cytometry (Thermo Fisher Scientific, Invitrogen, catalog number: MF48005 )
PE anti-mouse CD206 (MMR) antibody (BioLegend, catalog number: 141705 )
DAPI (Vector Laboratories, catalog number: H-1200 )
Anti-mouse E-Cadherin antibody (BD, Pharmingen^TM, catalog number: 610404 )
Donkey anti-mouse (Alexa Flour 594) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-21203 )
Live cell tracker CMFDA (Thermo Fisher Scientific, Invitrogen^TM, catalog number: C2925 )
EGFP (abm, catalog number: LV011-a )
Na₂HPO₄
NaCl
KH₂PO₄
KCl
Corning Matrigel (Basement membrane matrix, Corning, catalog number: 356234 )
Tween 20 (Sigma-Aldrich, catalog number: P7949-500ML )
Paraformaldehyde (Sigma-Aldrich, catalog number: 16005 )
PBS (see Recipes)
PBST (see Recipes)
AC buffer (0.5% BSA) (see Recipes)
2% Matrigel (see Recipes)
3.7% paraformaldehyde (see Recipes)

Equipment

Safety cabinet
Pipette-aid
Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
Water bath set at 37 °C
Humidified cell culture incubator set to 37 °C and 5% CO₂
Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss, model: Axiovert 200 )
Upright microscope with 10x objective
Cell sorter and the scale (BD, model: FACSAria^TM II ); sorting at a rate of 80,000 cell/h

Software

Quantitation of the average distance between the geometrical center of nuclei of adjacent cells can be measured using Openlab3 software (Improvision, Lexington, MA) or other commercially available image analysis software
SAS software (version 9.1.4, SAS Institute, Cary, NC)

Procedure

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中文翻译

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如何引用

Long, L., Yin, M. and Min, W. (2018). 3D Co-culture System of Tumor-associated Macrophages and Ovarian Cancer Cells. Bio-protocol 8(8): e2815. DOI: 10.21769/BioProtoc.2815.