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Recherche en cancérologie

부인과 및 유방암 종양에서 암 줄기 세포 구체 획득

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

이 방법론의 목적은 유세포분석및 서양세포분석과 서양계의 기능성 분석및 현상형 특성화를 사용하여, 구형 프로토콜을 사용하여 암 세포주 및 원발성 인간 종양 샘플에서 암 줄기세포(CSC)를 식별하는 것입니다. 얼룩.

Abstract

암 줄기 세포 (CSC)는 종양 발생, 치료 및 재발성 질병에 대한 저항성을 담당하는 자가 재생 및 가소성을 가진 작은 집단입니다. 이 모집단은 표면 마커, 효소 활성 및 기능 적 프로파일로 식별 할 수 있습니다. 이러한 접근법은 현상형 이질성과 CSC 가소성으로 인해 제한적입니다. 여기에서, 우리는 유방과 부인과 암에서 CSC 구체를 얻기 위하여 구체 형성 프로토콜을, 기능적인 성질, CSC 마커 및 단백질 발현을 평가하기 위하여 업데이트합니다. 구체는 현탁액 배양에서 낮은 밀도로 단일 세포 파종으로 수득되며, 반고체 메틸셀룰로오스 배지를 사용하여 이동 및 응집체를 피합니다. 이 수익성 있는 프로토콜은 암 세포주뿐만 아니라 1 차적인 종양에서 또한 이용될 수 있습니다. 종양 미세 환경, 특히 CSC 틈새 를 모방하는 것으로 생각되는 삼차원 비 부착 현탁액 배양은 CSC 신호링을 보장하기 위해 표피 성장 인자 및 기본 섬유아세포 성장 인자로 보충됩니다. CSC의 강력한 식별을 목표로 기능및 자형학적 평가를 결합한 보완적인 접근 방식을 제안합니다. 구 형성 용량, 자체 재생 및 구 투영 영역은 CSC 기능적 특성을 설정합니다. 부가적으로, 특성화는 ALDH를 고려한 CD44+/CD24-및 CD133 및 웨스턴 블롯으로 표현된 마커의 유세포분석 평가를 포함한다. 제시된 프로토콜은 또한 번역 연구에 유용한 샘플 소화 절차에 따라 1 차적인 종양 견본을 위해 최적화되었습니다.

Introduction

암 인구는 이질적인, 다른 형태를 제시 하는 세포, 증식 및 침략 능력, 차등 유전자 발현으로 인해. 이들 세포 들 중, 소수 집단은 암 줄기 세포 (CSC)1이라는이름의 존재, 이는 자기 갱신을위한 능력을 가지고, 1 차 종양 틈새의 이질성을 재차화하고 동압 조절에 적절하게 반응하지 않는 비정상적인 분화 선전자를 생산2. CSC 특성은 화학요법에 대한 종양발생성 또는 내성과 같은 사건과의 연관성을 감안할 때 임상 실습에서 직접 번역될 수있다 3. CSC의 식별은 표면 마커의 막힘, CSC 분화의 촉진, CSC 신호 경로 구성 요소의 차단, 틈새 파괴 및 후성 유전학 적 메커니즘을 포함 할 수있는 표적 치료의 개발로 이어질 수 있습니다4.

CSC의 분리는 세포 선및 1 차적인 종양의 견본에서5,6,7,8에서수행되었습니다. CSC에 대해 기재된 기능적 프로파일은 투압 용량, 측 군수 및 종양구 형성9를포함한다. CD44고/CD24저표현형은 유방 CSC와 일관되게 연관되어 왔으며, 이는 생체 내에서 종양발생이 입증되었으며 이미 중피전도5,10에상피와 연관되어 있다. 높은 ALDH 활성은 또한 고형종양(11)의여러 유형에서 중간엽 전이(EMT)에 대한 줄기 및 상피와 연관되어 있다. ALDH 발현은 화학요법 및 체외에서 CSC 표현형에 대한 내성과 연관되어 있다12,13,14,15,16. 몇몇 다른 마커들은 표 1에기재된 바와 같이 CD133, CD49f, ITGA6, CD1663,4 및 기타와 같은 상이한 유형의 종양에서 CSC 특성에 연결되었다.

종양구는 CSC의 연구 및 확장을 위한 3차원 모델로 구성됩니다. 이 모델에서, 세포주및 혈액 또는 종양 샘플로부터의 세포 현탁액은 성장 인자, 즉 표피 성장 인자(EGF) 및 기본 섬유아세포 성장 인자(bFGF)로 보충된 배지에서 배양되며, 태아 소 혈청 없이 비부착 조건에서17. 세포 접착의 억제는 분화세포의 아노이키스에 의한 사망을초래한다(18). 구체는 분리된 세포의 클론 성장으로부터 유래된다. 이를 위해, 세포는 세포 융합 및응집(19)을피하기 위해 저밀도로 분포된다. 또 다른 전략은 반고체 메틸셀룰로오스20의사용을 포함한다.

구형 프로토콜은 CSC 격리 및 확장에서 인기를 얻었으며, 시간 및 비용 및 기술, 수익성 및 재현 가능한 이유21,22. 구형이 CSC를 반영하는 정도에 대한 일부 예비에도 불구하고, 줄기 세포가 네이티브 미세 환경21과유사한 특성 표현형을 가진 비 부착 조건에서 성장하는 경향이 있다. 고형 종양에서 CSC를 격리하는 데 사용할 수있는 방법 중 어느 것도 완전한 효율성을 가지고 있지 않으며, 방법론과 마커의 보다 구체적인 마커 또는 조합을 개발하는 것의 중요성을 강조합니다.

이 프로토콜에서는 비부착 조건에서 단세포 성장원리와 차별화된 표현형을 생성하는 능력으로 CSC의 격리를 구형 프로토콜로 자세히 설명합니다. 이 절차의 개략적 표현은 그림 1에표시됩니다. 우리는 또한 CSC를 위한 표면 마커 및 ALDH 표현을 가진 특성화를 기술합니다, 유방과 부인과 종양 세포 선 및 1 차적인 종양의 견본을 위해 둘 다.

Protocol

이 프로토콜은 코임브라 병원과 대학 센터 (CHUC) 종양 은행의 윤리 지침을 준수 수행하고, 건강에 대한 CHUC의 윤리위원회와 포르투갈 국가 데이터 보호위원회에 의해 승인되었다. 1. 연속 세포 배양에서 구체 형성 프로토콜 및 파생 된 부착 인구 참고: 엄격한 멸균 조건하에서 모든 절차를 수행하십시오. 비 부착 현탁액 배양 플라스크 또는 플레이트…

Representative Results

구체 형성 프로토콜은 여러 자궁내막 및 유방암 세포주로부터 현탁액에서 또는인간 종양 샘플로부터 조직의 부드러운 효소 소화 후에 구형 콜로니를 수득할 수 있게한다(그림 2E). 두 경우 모두, 도금 후 며칠, 서스펜션의 단일 클론 구형 콜로니가 얻어진다. 자궁내막 및 유방암 구체 모두 기원의 세포주와 유사한 형태를 가진 ?…

Discussion

이 프로토콜은 암 세포주 및 1 차적인 인간 견본에게서 종양구를 장악하기 위하여 접근을 상세히 설명합니다. 종양구는 줄기세포와 같은 성질을 가진 하위 집단에서 풍부하게 된다36. CSC에서의 이러한 농축은 분화된 세포가기판(37)에부착되는 동안 앵커리지없는 환경에서의 생존력에 의존한다. 현탁액을 부과하는 낮은 부착 환경에서 종양 세포의 1 차 도금이 CSC…

Divulgations

The authors have nothing to disclose.

Acknowledgements

이 연구 결과는 2016 연구 상을 통해 그리고 CIMAGO에 의해 부인과의 포르투갈 사회에 의해 투자되었습니다. Cnc. IBILI는 포르투갈 과학 기술 재단(UID/NEU/04539/2013)을 통해 지원되며 FED-COMPETE(POCI-01-01-0145-FEDER-007440)가 공동 후원합니다. Coimbra 병원 과 대학 센터 (CHUC) 종양 은행, 건강에 대한 CHUC의 윤리위원회와 포르투갈 국가 데이터 보호위원회에 의해 승인, 기관의 부인과 서비스에서 다음 환자의 자궁 내막 샘플의 소스였다. 도 1은 www.servier.com에서 구할 수 있는 세르비에 메디컬 아트를 사용하여 제작되었다.

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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