Imaging-とフローサイトメトリーベースのセル位置の解析と3Dメラノーマスフェロイドにおける細胞周期
Summary
We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.
Abstract
Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.
Introduction
多3Dスフェロイドが数十年のための腫瘍モデルとして知られている、しかし、それは彼らが多くの固形癌のためのin vitroモデルとして、より一般的に使用されるようになったことはごく最近です。彼らはますます複雑、高価で時間のかかる生体内モデルで 、シンプルで低コストの2D単層モデル1-6との間の中間のような高スループットの薬物発見スクリーニングにおいて使用されています。 2D培養における研究は、多くの場合、 生体内で複製することができません。癌の多くの種類の回転楕円体モデルは、増殖特性、薬剤感受性、薬剤の浸透、細胞-細胞相互作用、固形腫瘍6-11 においてインビボで見られる酸素や栄養素および壊死の開発の制限された利用可能性を模倣することができます。スフェロイドは、回転楕円体7の周囲に壊死性コア、コアを取り囲む逮捕静止またはG1領域、および増殖細胞を開発しています。これらの領域の開発細胞密度、増殖速度および回転楕円体12の大きさに応じて変えることができます。これらの異なるサブ領域で見られる細胞の異質性は、癌治療抵抗13,14に寄与し得ると仮定されています。したがって、これらの領域内の細胞を分析する能力は、個別の腫瘍薬物応答を理解するために重要です。
緑（あざみグリーン- AG）は、細胞周期15の異なる段階に分解されるCDT1とGemininの蛍光タグ付け、 -蛍光ユビキチン化、細胞周期のインジケータ（FUCCI）システムは、赤（KO Kusabiraオレンジ）に基づいています。従って、細胞核は、初期のS内の黄色、G1の赤とS / G2 / M期で緑に表示されます。ここでは二つの相補的な方法を記載している両方の細胞がG1逮捕中央または外側proliferatinに存在するかどうかを決定するために画像処理ソフトウェアまたは染料拡散フローサイトメトリーアッセイの使用に伴って、細胞周期を特定するためにFUCCIを用いグラムリング、及び回転楕円体の端部から個々の細胞の距離。これらの方法は、我々は回転楕円体の中心にあるおよび/または標的療法の存在下での低酸素領域におけるメラノーマ細胞が長期間G1停止のままにすることが可能であり、再できることを実証した我々の以前の出版物、に開発されましたより有利な条件は7を生じたとき、細胞周期を入力してください。
Protocol
Representative Results
Discussion
半自動画像解析は、回転楕円体の内側G1逮捕領域を同定し、そして外層の増殖。この方法は、これらの異なる領域で（免疫蛍光により）、細胞周期が、マーカー発現しないだけの変化を識別するために、光学部を使用して、ライブスフェロイドに、または一定の回転楕円体のセクションで使用され、細胞死、または細胞の形態であってもよいです。異なる回転楕円体領域内の細胞の運動性も?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Ms. Danae Sharp and Ms. Sheena Daignault for technical assistance. We thank Dr. Atsushi Miyawaki, RIKEN, Wako-city, Japan, for providing the FUCCI constructs, Dr. Meenhard Herlyn and Ms. Patricia Brafford, The Wistar Institute, Philadelphia, for providing cell lines, the Imaging and Flow Cytometry Facility at the Centenary Institute for outstanding technical support. We thank Mr. Chris Johnson and Dr. Andrew Barlow for Volocity software technical support. N.K.H. is a Cameron fellow of the Melanoma and Skin Cancer Research Institute, Australia. K.A.B. is a fellow of the Cancer Institute New South Wales (13/ECF/1-39). W.W. is a fellow of the Cancer Institute New South Wales (11/CDF/3-39). This work was supported by project grants RG 09-08 and RG 13-06 (Cancer Council New South Wales), 570778 and 1051996 (Priority-driven collaborative cancer research scheme/Cancer Australia/Cure Cancer Australia Foundation), 08/RFG/1-27 (Cancer Institute New South Wales), and APP1003637 and APP1084893 (National Health and Medical Research Council).
Materials
| Hoechst 33342 | Life Technologies | H3570 | |
| agarose low melting point | Life Technologies | 16520-050 | For sectioning |
| noble agar | Sigma | A5431 | For making spheroids |
| agarose for spheroids | Fisher Scientific | BP1356-100 | For making spheroids |
| 0.05% trypsin/EDTA | Life Technologies | 25300-054 | |
| HBSS | Life Technologies | 14175-103 | |
| 10% formalin | Sigma | HT5014-1CS | CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do not breath fumes (open in a fume cupboard). |
| live/dead near IR | Life Technologies | L10119 | |
| vibratome | Technical Products International, Inc | ||
| coulture cup | Thermo-Fisher Scientific | SIE936 | Mold for sectioning spheroids |
| hemocytometer | Sigma | Z359629 | |
| 96-well tissue culture plate | Invitro | FAL353072 | |
| collagenase | Sigma | C5138 | |
| confocal microscope | Leica | TCS SP5 | |
| Flow cytometer analyser | Becton Dickinson | LSRFortessa | |
| volocity | PerkinElmer | Imaging software | |
| flowjo | Tree Star | Flow cytometry software | |
| Vaccuum grease | Sigma | Z273554 | |
| Mounting media | Vector Laboratories | H1000 | |
| FUCCI (commercial constructs) | Life Technologies | P36238 | Transient transfection only |
| Cell strainer 70 um | In Vitro | FAL352350 | |
| Round bottom 5 mL tubes (sterile) | In Vitro | FAL352003 | |
| Round bottom 5 mL tubes (non-sterile) | In Vitro | FAL352008 |
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