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Ricerca sul cancro

Jinekolojik ve Meme Kanseri Tümörlerinden Kanser Kök Hücre Kürelerinin Alınması

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

Bu metodolojinin amacı, kanser hücre hatlarındaki kanser kök hücrelerini (CSC) ve primer insan tümör örneklerini küre oluşturma protokolü ile sağlam bir şekilde, fonksiyonel tahliller ve akış sitometrisi ve Batı ile henotypik karakterizasyon kullanarak tanımlamaktır. Leke.

Abstract

Kanser kök hücreleri (CSC) tümörigenez, tedaviye direnç ve tekrarlayan hastalıklardan sorumlu kendi kendini yenileme ve plastisite ile küçük bir popülasyondur. Bu popülasyon yüzey belirteçleri, enzimatik aktivite ve fonksiyonel profil ile tanımlanabilir. Bu yaklaşımlar, henotibik heterojenlik ve CSC plastisite nedeniyle sınırlıdır. Burada, meme ve jinekolojik kanserlerden CSC küreleri elde etmek, fonksiyonel özellikleri, CSC belirteçleri ve protein ekspresyonu değerlendirmek için küre oluşturan protokolü güncelliyoruz. Küreler, süspansiyon kültüründe düşük yoğunlukta tek hücreli tohumlama ile, göç ve agregaları önlemek için yarı katı metilselüloz orta kullanılarak elde edilir. Bu karlı protokol kanser hücre hatlarında değil, primer tümörlerde de kullanılabilir. Tümör mikroçevresini taklit ettiği düşünülen üç boyutlu non-yapışık süspansiyon kültürü, özellikle CSC-niş, epidermal büyüme faktörü ve temel fibroblast büyüme faktörü ile csc sinyalizasyon sağlamak için desteklenmektedir. CSC’nin sağlam bir şekilde tanımlanmasını hedefleyen, fonksiyonel ve henotipik değerlendirmeyi birleştiren tamamlayıcı bir yaklaşım öneriyoruz. Küre oluşturma kapasitesi, kendini yenileme ve küre projeksiyon alanı CSC fonksiyonel özellikleri oluşturur. Ayrıca, karakterizasyon, ALDH göz önünde bulundurularak CD44+/CD24– ve CD133 ve Western blot ile temsil edilen işaretçilerin akış sitometri değerlendirmesini içerir. Sunulan protokol ayrıca, çevirisel araştırmalar için yararlı olan bir örnek sindirim prosedürü nden sonra primer tümör örnekleri için optimize edildi.

Introduction

Kanser popülasyonları heterojendir, farklı morfolojiler, proliferasyon ve invazyon kapasitesi sunan hücreler, diferansiyel gen ekspresyonuna bağlı olarak. Bu hücreler arasında, bir azınlık popülasyon kanser kök hücreleri adlı var (CSC)1, kendi kendini yenileme kapasitesine sahip, primer tümör niş heterojenite recapitulating ve homeostatik kontrollere yeterince yanıt vermez anormal ayırt edici ataları üreten2. CSC özellikleri doğrudan klinik uygulamada tercüme edilebilir, olaylar ile ilişki göz önüne alındığında, tümörijenite veya kemoterapi direnci gibi3. CSC’nin tanımlanması, yüzey belirteçlerinin tıkanmasını, CSC farklılaşmasının teşvikini, CSC sinyal yol bileşenlerinin engellenmesini, niş yıkımını ve epigenetik mekanizmaları içerebilir hedefli tedavilerin geliştirilmesine yol açabilir4.

CSC izolasyonu hücre hatlarında ve primer tümör örneklerinde5,6,7,8olarak gerçekleştirilmiştir. CSC için tanımlanan fonksiyonel profil klojenik kapasite, yan popülasyon ve tümöroferoluşumunuiçerir 9 . CD44yüksek/ CD24düşük fenotip sürekli meme CSC ile ilişkili olmuştur, hangi in vivo tümörijenik olduğu kanıtlanmıştır ve zaten mezenkimal geçiş epitel ile ilişkili olmuştur5,10. Yüksek ALDH aktivitesi de solid tümörlerin çeşitli tiplerinde kalsuk ve mezenkimal geçiş (EMT) epitelyal ile ilişkili olmuştur11. ALDH ekspresyonu kemoterapiye direnç ve CSC fenotip in vitro12,13,14,15,16ile ilişkili olmuştur. Cd133, CD49f, ITGA6, CD1663,4 ve diğerleri gibi farklı tümör türlerinde CSC özelliklerine eklenmiştir.

Tümör küreleri çalışma ve CSC genişlemesi için üç boyutlu bir model oluşur. Bu modelde, hücre hatları ve kan veya tümör örneklerinden hücre süspansiyonları büyüme faktörleri ile desteklenen bir ortamda yetiştirilmektedir, yani epidermal büyüme faktörü (EGF) ve temel fibroblast büyüme faktörü (bFGF), fetal sığır serum olmadan ve non-adherent koşullarda17. Hücre adezyonunun inhibisyonu, farklılaşmış hücrelerin anoikis tarafından ölümle sonuçlanır18. Küreler izole bir hücrenin klonal büyüme türetilmiştir. Bu amaçla, hücreler hücre füzyonu ve toplama önlemek için düşük yoğunlukta dağıtılır19. Başka bir strateji yarı katı metilselüloz kullanımını içerir20.

Küre oluşturan protokol CSC izolasyon ve genişleme popülerlik kazandı, zaman ve maliyet ve teknik nedeniyle, karlı, ve tekrarlanabilir nedenlerle21,22. Küre oluşumunun CSC’yi ne ölçüde yansıttığına dair bazı rezervlere rağmen, kök hücrelerin doğal mikroçevreye benzeyen karakteristik fenotip ile yapışık olmayan koşullarda büyüme eğilimi vardır21. CSC’nin katı tümörlerden izole edilmesi için kullanılan yöntemlerin hiçbiri tam bir verime sahip değildir ve daha spesifik belirteçler veya metodoloji ve belirteç kombinasyonları geliştirmenin önemini vurgulamaktadır.

Bu protokolde, CSC’nin küre oluşturan protokolle izolasyonu, yapışık olmayan koşullarda tek hücreli büyüme prensibi ve farklılaştırılmış fenotip üretme kapasitesi ile ayrıntılarıyla açıklanmaktadır. Bu yordamın şematik bir gösterimi Şekil 1’detemsil edilir. Ayrıca meme ve jinekolojik tümör hücreleri hatları ve primer tümör örnekleri için, csc için yüzey belirteçleri ve ALDH ekspresyonu ile karakterizasyonu açıklıyoruz.

Protocol

Bu protokol Coimbra Hastanesi ve Universitary Center (CHUC) Tümör Bankası’nın etik kurallarına uygun olarak gerçekleştirildi ve CHUC’nin Sağlık Etik Komitesi ve Portekiz Ulusal Veri Koruma Komisyonu tarafından onaylandı. 1. Sürekli Hücre Kültürlerinden Küre Oluşturan Protokol ve Türemiş Yapışık Popülasyonlar NOT: Tüm prosedürleri katı steril koşullar altında gerçekleştirin. Büyüme yüzeyini poli (2-hidroksietil-metakrilat (p…

Representative Results

Küre oluşturma protokolü, küresel kolonilerin birkaç endometriyal ve meme kanseri hücre hattından süspansiyon içinde(Şekil 2A)veya insan tümör örneklerinden doku ların nazik enzimatik sindiriminden sonra elde edilmesine olanak sağlar (Şekil 2E). Her iki durumda da kaplamadan birkaç gün sonra süspansiyonlu monoklonal küresel koloniler elde edilir. Hem endometrial hem de meme kanseri küreleri, hücre nin menş…

Discussion

Bu protokol, kanser hücre hatları ve birincil insan örneklerinden tümör küreleri elde etmek için bir yaklaşım ayrıntıları. Tümörküreler kök hücre benzeri özelliklere sahip bir alt popülasyonda zenginleştirilmiştir36. CSC’deki bu zenginleştirme, ankrajsız bir ortamda canlılığa bağımlıyken, farklılaşmış hücreler37. Süspansiyon uyguluyor düşük bir yapışma ortamında tümör hücrelerinin birincil kaplama olarak kendi kendine yenileme (k?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Bu çalışma Portekiz Jinekoloji Derneği tarafından 2016 Araştırma Ödülü ve CIMAGO tarafından finanse edilmiştir. Cnc. IBILI, Portekiz Bilim ve Teknoloji Vakfı (UID/NEU/04539/2013) ve FEDER-COMPETE (POCI-01-0145-FEDER-007440) tarafından finanse edilmektedir. CHUC Etik Sağlık Komitesi ve Portekiz Ulusal Veri Koruma Komisyonu tarafından onaylanan Coimbra Hastanesi ve Universitary Center (CHUC) Tümör Bankası, kurumun Jinekoloji Servisi’nde takip edilen hastaların endometrial örneklerinin kaynağı oldu. Şekil 1 Servier Tıp Sanatı kullanılarak üretildi, www.servier.com.

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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Keywords: 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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