JoVE Journal > Cancer Research
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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암 연구학

婦人科腫瘍および乳がん腫瘍から癌幹細胞球体を取得する

Published: March 01, 2020
doi:
10.3791/60022
, , , , , , ,
1Institute of Biophysics, Faculty of Medicine,University of Coimbra, 2Coimbra Institute for Clinical and Biomedical Research (iCBR), area of Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine,University of Coimbra, 3CNC.IBILI/Center for Innovative Biomedicine and Biotechnology (CIBB),University of Coimbra, 4Clinical Academic Center of Coimbra (CACC), 5Universitary Clinic of Gynecology, Faculty of Medicine,University of Coimbra, 6Gynecology A Service,Coimbra Hospital and Universitary Center, 7Institute of Pharmacology & Experimental Therapeutics, Faculty of Medicine,University of Coimbra, 8Institute of Experimental Pathology, Faculty of Medicine,University of Coimbra, 9Cytometry Operational Management Unit, Clinical Pathology Service,Coimbra Hospital and Universitary Center, 10Polytechnic Institute of Coimbra, ESTESC-Coimbra Health School,Laboratory Biomedical Sciences

Summary

この方法論の目的は、癌細胞株および原発ヒト腫瘍試料中の癌幹細胞(CSC)を、細胞細胞の機能性アッセイと表現型特性評価を用いて堅牢に同定することである。しみ。

Abstract

がん幹細胞(CSC)は、腫瘍の発生、治療に対する耐性および再発性疾患の原因となる自己再生性および可塑性を有する小さな集団である。この集団は、表面マーカー、酵素活性および機能的プロファイルによって同定することができる。これらのアプローチは、その他、異種性とCSC可塑性のため、制限されています。ここでは、球体形成プロトコルを更新して、乳癌および婦人科癌からCSC球体を取得し、機能的特性、CSCマーカーおよびタンパク質発現を評価する。球体は、遊入および凝集を回避するために半固体メチルセルロース培地を用いて、懸濁培養における低密度での単細胞播種で得られる。この有益なプロトコルは、癌細胞株だけでなく、原発性腫瘍でも使用することができる。三次元非接着性懸濁液培養は、腫瘍微小環境、特にCSCニッチを模倣すると考え、CSCシグナル伝達を確実にするために表皮成長因子および塩基性線維芽細胞増殖因子を補う。CSCの強固な同定を目指し、機能的評価と表型評価を組み合わせた補完的なアプローチを提案する。球体形成能力、自己更新、および球投影領域は、CSC の機能特性を確立します。さらに、特性評価は、マーカーのフローサイトメトリー評価を含み、CD44+/CD24-およびCD133で表され、ALDHを考慮してウェスタンブロットを含む。提示されたプロトコルは、原発性腫瘍サンプルにも最適化され、サンプル消化手順に従い、翻訳研究に有用であった。

Introduction

癌集団は異種であり、細胞は異なる形態、増殖および浸潤能力を示し、遺伝子発現の変化による。これらの細胞の中には、自立能力を有する癌幹細胞(CSC)1という名前の少数集団が存在し、原発性腫瘍ニッチの異質性を再現し、恒恒恒転移制御に適切に反応しない異常に分化した前駆体を作り出す2。CSC特性は、腫瘍原性または化学療法に対する耐性などの事象との関連を考えると、臨床現場で直接翻訳することができる3.CSCの同定は、表面マーカーの閉塞、CSC分化の促進、CSCシグナル伝達経路成分の遮断、ニッチ破壊、およびエピジェネティック機構4を含む標的療法の開発につながる可能性がある。

CSCの単離は、細胞ラインおよび原発腫瘍のサンプル5、6、7、8で行われている。CSCについて記載された機能的プロファイルは、クロノ原性、側集団および腫瘍球形成9を含む。CD44高/CD24低表現型は乳房CSCと一貫して関連付けられており、これは生体内で腫瘍形成であることが証明されており、すでに間葉転移5、10に上皮と関連している。高いALDH活性はまた、いくつかのタイプの固形腫瘍11における間葉転移(EMT)に対する茎および上皮と関連している。ALDH発現は、インビトロ12におけるCSC表現型に対する化学療法およびCSC表現型に対する耐性と関連付けられてきたが、13、14、15、16。表1に記載されているように、CD133、CD49f、ITGA6、CD1663、4など、異なるタイプの腫瘍におけるCSC特性にリンクされている他のいくつかのマーカーが挙げられております。

腫瘍球は、CSCの研究と拡張のための3次元モデルで構成されています。このモデルでは、細胞株および血液または腫瘍試料からの細胞懸濁液を、成長因子を添加した培地、すなわち表皮成長因子(EGF)および塩基性線維芽細胞増殖因子(bFGF)で培養し、ウシ胎児血清および非付着条件下で17。細胞接着の阻害は、分化細胞18のアノイキスによる死をもたらす。球は、分離された細胞のクローン成長から導出されます。この目的のために、細胞は細胞融合および凝集19を避けるために低密度で分布する。別の戦略は、半固体メチルセルロース20の使用を含む。

球体形成プロトコルは、時間とコストと技術的、収益性、および再現可能な理由21、22のためにCSCの分離と拡張で人気を集めました。どの球形成がCSCを反映する程度に関するいくつかの埋蔵量にもかかわらず、天然の微小環境21に似た特徴的な表現型を有する非接着条件で成長する幹細胞の傾向がある。固形腫瘍からのCSCの単離に利用可能な方法のいずれも完全な効率を持っておらず、より具体的なマーカーまたは方法論およびマーカーの組み合わせを開発することの重要性を強調する。

このプロトコルでは、非粘着性条件における単細胞増殖の原理と分化表現型を生成する能力を持つ、球形成プロトコルを用いたCSCの分離について詳述する。この手順の概略図を図1に示します。また、乳房および婦人科腫瘍細胞のラインと原発性腫瘍のサンプルの両方に対するCSCの表面マーカーおよびALDH発現を用いた特性評価についても述べている。

Protocol

このプロトコルは、コインブラ病院および大学センター(CHUC)腫瘍銀行の倫理ガイドラインに準拠して実施され、CHUCの健康倫理委員会とポルトガル国家データ保護委員会によって承認されました。 1. 連続細胞培養による球体形成プロトコルと導付性集団 メモ:厳格な無菌条件下ですべての手順を実行します。 ポリ(2-ヒドロキセチルメタクリル?…

Representative Results

球体形成プロトコルにより、球状コロニーを、いくつかの子宮内膜および乳癌細胞株(図2A)またはヒト腫瘍サンプルからの組織の穏やかな酵素消化後から懸濁液中で得ることができる(図2E)。いずれの場合も、めっきの数日後に、懸濁液中のモノクローナル球状コロニーが得られる。子宮内膜および乳癌球体の両方が、細胞?…

Discussion

このプロトコルは、がん細胞株および一次ヒトサンプルから腫瘍球を得るためのアプローチを詳述している。腫瘍球は、幹細胞様特性を有するサブ集団に富む36.CSCにおけるこの富化は、分化細胞が基材37への接着に依存している間、アンカレッジフリー環境での生存率に依存する。サスペンションを課す低い付着環境での腫瘍細胞の原発めっきはCSC自身の?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

この研究は、2016年の研究賞を通じてポルトガル婦人科学会とCIMAGOによって資金提供されました。Cnc。IBILIは、ポルトガル科学技術財団(UID/NEU/04539/2013)を通じて支援され、FEDER-COMPETE(POCI-01-0145-FEDER-007440)が共同出資しています。CHUCの健康倫理委員会とポルトガル国家データ保護委員会によって承認されたコインブラ病院と大学センター(CHUC)腫瘍銀行は、同機関の婦人科サービスに続く患者の子宮内膜サンプルの供給源でした。図1は、www.servier.comから入手可能なサーヴィエ医療アートを使用して製造された。

Materials

Absolute ethanol Merck Millipore 100983
Accutase Gibco A1110501 StemPro Accutas Cell Dissociation Reagent
ALDH antibody Santa Cruz Biotechnology SC166362
Annexin V FITC BD Biosciences 556547
Antibiotic antimycotic solution Sigma A5955
BCA assay Thermo Scientific 23225 Pierce BCA Protein Assay Kit
Bovine serum albumin Sigma A9418
CD133 antibody Miteny Biotec 293C3-APC Allophycocyanin (APC)
CD24 antibody BD Biosciences 658331 Allophycocyanin-H7 (APC-H7)
CD44 antibody Biolegend 103020 Pacific Blue (PB)
Cell strainer BD Falcon 352340 40 µM
Collagenase, type IV Gibco 17104-019
cOmplete Mini Roche 118 361 700 0
Dithiothreitol Sigma 43815
DMEM-F12 Sigma D8900
DNAse I Roche 11284932001
ECC-1 ATCC CRL-2923 Human endometrium adenocarcinoma cell line
Epidermal growth factor Sigma E9644
Fibroblast growth factor basic Sigma F0291
Haemocytometer VWR HERE1080339
HCC1806 ATCC CRL-2335 Human mammary squamous cell carcinoma cell line
Insulin, transferrin, selenium Solution Gibco 41400045
MCF7 ATCC HTB-22 Human mammary adenocarcinoma cell line
Methylcellulose AlfaAesar 45490
NaCl JMGS 37040005002212
Poly(2-hydroxyethyl-methacrylate Sigma P3932
Putrescine Sigma P7505
RL95-2 ATCC CRL-1671 Human endometrium carcinoma cell line
Sodium deoxycholic acid JMS EINECS 206-132-7
Sodium dodecyl sulfate Sigma 436143
Tris JMGS 20360000BP152112
Triton-X 100 Merck 108603
Trypan blue Sigma T8154
Trypsin-EDTA Sigma T4049
��-actin antibody Sigma A5316
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Cancer Stem CellsCancer Stem Cell SpheresSphere-forming AssayPolyHEMANon-adherent Suspension CultureEpidermal Growth FactorBasic Fibroblast Growth FactorSphere-forming CapacityAdherent SpheresHemocytometerGynecological CancerBreast Cancer
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Laranjo, M., Carvalho, M. J., Serambeque, B., Alves, A., Marto, C. M., Silva, I., Paiva, A., Botelho, M. F. Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors. J. Vis. Exp. (157), e60022, doi:10.3791/60022 (2020).
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