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Abstract
Introduction
Protocol
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Pesquisa do Câncer

Het verkrijgen van kanker stamcelbollen van gynaecologische en borstkankertumoren

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

Het doel van deze methodologie is om kankercellen (CSC) te identificeren in kankercellijnen en primaire menselijke tumormonsters met het bolvormende protocol, op een robuuste manier, met behulp van functionele testen en fenotypische karakterisering met stroomcytometrie en westerse Vlek.

Abstract

Kankercellen (CSC) zijn een kleine populatie met zelfvernieuwing en plasticiteit die verantwoordelijk zijn voor tumorigenese, resistentie tegen behandeling en terugkerende ziekte. Deze populatie kan worden geïdentificeerd door oppervlaktemarkers, enzymatische activiteit en een functioneel profiel. Deze benaderingen per se zijn beperkt, als gevolg van fenotypische heterogeniteit en CSC plasticiteit. Hier werken we het bolvormende protocol bij om CSC-bollen te verkrijgen van borst- en gynaecologische kankers, waarbij functionele eigenschappen, CSC-markers en eiwitexpressie worden beoordeeld. De bollen worden verkregen met eencellige zaaien bij lage dichtheid in de suspensiecultuur, met behulp van een halfvast methylcellulosemedium om migratie en aggregaten te voorkomen. Dit winstgevende protocol kan worden gebruikt in kankercellen, maar ook in primaire tumoren. De driedimensionale niet-aanhangende suspensie cultuur dacht aan de tumor micro-omgeving na te bootsen, met name de CSC-niche, wordt aangevuld met epidermale groeifactor en fundamentele fibroblast groeifactor om CSC signalering te waarborgen. Met het oog op een robuuste identificatie van CSC stellen we een complementaire aanpak voor, waarbij functionele en fenotypische evaluatie wordt gecombineerd. Gebiedsvormend vermogen, zelfvernieuwing en sfeerprojectiegebied vestigen csc-functionele eigenschappen. Daarnaast omvat karakterisering de evaluatie van de stroomcytometrie van de markers, vertegenwoordigd door CD44+/CD24– en CD133, en Western blot, gezien ALDH. Het gepresenteerde protocol werd ook geoptimaliseerd voor primaire tumormonsters, na een steekproefverteringsprocedure, nuttig voor translationeel onderzoek.

Introduction

De bevolking van kanker is heterogeen, met cellen die verschillende morfologieën, proliferatie en invasiecapaciteit, toe te schrijven aan differentiële genuitdrukking voorstellen. Onder deze cellen bestaat een minderheidspopulatie genaamd kankercellen (CSC)1, die de capaciteit hebben voor zelfvernieuwing, het recapituleren van de heterogeniteit van de primaire tumorniche en het produceren van afwijkend differentiërende voorlopers die niet adequaat reageren op homeostatische controles2. CSC-eigenschappen kunnen direct worden vertaald in de klinische praktijk, gezien de associatie met gebeurtenissen, zoals tumorigeniciteit of resistentie tegen chemotherapie3. De identificatie van CSC kan leiden tot de ontwikkeling van gerichte therapieën die blokkade van oppervlaktemarkeringen, bevordering van CSC-differentiatie, blokkering van CSC-signaleringspadcomponenten, nichevernietiging en epigenetische mechanismen4kunnen omvatten.

De isolatie van CSC is uitgevoerd in cellenlijnen en in monsters van primaire tumoren5,6,7,8. Het functionele profiel beschreven voor CSC omvat clonogene capaciteit, zijbevolking en tumorosfeer vorming9. De CD44hoge/ CD24lage fenotype is consequent geassocieerd met borst CSC, die heeft bewezen tumorigeen in vivo en is al geassocieerd met epitheel aan mesenchymale overgang5,10. Hoge ALDH activiteit is ook geassocieerd met stamness en epitheel aan mesenchymale overgang (EMT) in verschillende soorten vaste tumoren11. ALDH-expressie is geassocieerd met resistentie tegen chemotherapie en csc fenotype in vitro12,13,14,15,16. Verschillende andere markers zijn gekoppeld aan CSC-eigenschappen in verschillende soorten tumoren, zoals CD133, CD49f, ITGA6, CD1663,4 en anderen, zoals beschreven in tabel 1.

De tumorsferen bestaan uit een driedimensionaal model voor de studie en uitbreiding van CSC. In dit model worden de celsuspensen van cellijnen en van bloed- of tumormonsters gekweekt in een medium aangevuld met groeifactoren, namelijk epidermale groeifactor (EFG) en basisfibroblastgroeifactor (bFGF), zonder foetaal runderserum en in niet-aanhangende omstandigheden17. Remming van celhechting resulteert in de dood door anoikis van gedifferentieerde cellen18. Bollen zijn afgeleid van de kloongroei van een geïsoleerde cel. Hiertoe worden de cellen bij een lage dichtheid verdeeld om celfusie en aggregatie te voorkomen19. Een andere strategie omvat het gebruik van halfvaste methylcellulose20.

Het gebied-vormende protocol won populariteit in CSC isolatie en uitbreiding, als gevolg van tijd en kosten en technische, winstgevende, en reproduceerbare redenen21,22. Ondanks enkele reserves over de mate waarvan de bolvorming CSC weerspiegelt, is er een neiging van stamcellen om te groeien in niet-aanhangende omstandigheden met het karakteristieke fenotype, dat lijkt op de inheemse micro-omgeving21. Geen van de methoden die beschikbaar zijn voor isolatie van CSC van vaste tumoren heeft volledige efficiëntie, met de nadruk op het belang van het ontwikkelen van meer specifieke markers of combinaties van methodologieën en markers.

In dit protocol beschrijven we de isolatie van CSC met het gebiedsvormende protocol, met het principe van eencellige groei in niet-aanhangende omstandigheden en het vermogen om een gedifferentieerd fenotype te produceren. Een schematische weergave van deze procedure is vertegenwoordigd in figuur 1. We beschrijven ook de karakterisering met oppervlaktemarkers en ALDH-expressie voor CSC, zowel voor borst- als gynaecologische tumorcellenlijnen en monsters van primaire tumoren.

Protocol

Dit protocol werd uitgevoerd in overeenstemming met de ethische richtlijnen van het Coimbra Hospital and Universitary Center (CHUC) Tumor Bank, en werd goedgekeurd door chuc’s Ethics Committee for Health en door de Portugese National Data Protection Commission. 1. Sfeervormingprotocol en afgeleide aanhangende populaties uit continue celculturen OPMERKING: Voer alle procedures uit onder strikte steriele omstandigheden. Bereiding van niet-aanhangende suspens…

Representative Results

Het bolvormende protocol maakt het mogelijk om sferische kolonies te verkrijgen in suspensie uit verschillende endometrium- en borstkankercellijnen(figuur 2A)of na zachte enzymatische vertering van weefsel uit menselijke tumormonsters(figuur 2E). In beide gevallen, een paar dagen na beplating, monoklonale bolvormige kolonies in suspensie worden verkregen. Zowel endometrium als borstkankerbollen geven aanleiding tot een celmonola…

Discussion

Dit protocol beschrijft een aanpak om tumorsferen te verkrijgen van kankercellijnen en primaire menselijke monsters. Tumorsferen zijn verrijkt in een subpopulatie met stamcelachtige eigenschappen36. Deze verrijking in CSC is afhankelijk van de levensvatbaarheid in een ankerplaatsvrije omgeving, terwijl gedifferentieerde cellen afhankelijk zijn van hechting aan een substraat37. Aangezien primaire beplating van tumorcellen in een lage hechtingsomgeving die schorsing oplegt, g…

Declarações

The authors have nothing to disclose.

Acknowledgements

Deze studie werd gefinancierd door de Portugese Vereniging van Gynaecologie via de Onderzoeksprijs 2016 en door CIMAGO. Cnc. IBILI wordt ondersteund door de Foundation for Science and Technology, Portugal (UID/NEU/04539/2013) en mede gefinancierd door FEDER-COMPETE (POCI-0145-FEDER-007440). Het Coimbra Hospital and Universitary Center (CHUC) Tumor Bank, goedgekeurd door chuc’s Ethics Committee for Health en door de Portugese National Data Protection Commission, was de bron van endometrium monsters van patiënten gevolgd bij de instelling Gynaecologie Service. Figuur 1 werd geproduceerd met behulp van Servier Medical Art, verkrijgbaar bij 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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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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