Beeldverwerking en flowcytometrie-gebaseerde analyse van de Cel standpunt en de celcyclus in 3D Melanoom Sferoïden
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
Multicellulaire sferoïden 3D zijn bekend als een tumormodel decennia, maar het is pas onlangs aan dat zij vaker gebruik als een in vitro model voor vele vaste kankers zijn gekomen. Ze worden steeds vaker gebruikt in high-throughput drug discovery schermen als intermediair tussen complexe, kostbare en tijdrovende in vivo modellen en de eenvoudige, goedkope 2D monolaag model 1-6. Studies in 2D cultuur zijn vaak niet in staat om te worden gerepliceerd in vivo. Bolvormige modellen van vele soorten kanker kunnen de groeikenmerken, drug gevoeligheid, geneesmiddelpenetratie, cel-cel interacties, beperkte beschikbaarheid van zuurstof en voedingsstoffen en ontwikkeling van necrose die wordt gezien in vivo bij vaste tumoren 6-11 nabootsen. Sferoïden ontwikkelen van een necrotische kern, een zwakke of G1 aangehouden regio rond de kern, en prolifererende cellen op de omtrek van de sferoïde 7. De ontwikkeling van deze regio'sis afhankelijk van de celdichtheid, proliferatiesnelheid en de omvang van de sferoïde 12. Er is verondersteld dat de cellulaire heterogeniteit waargenomen in de verschillende sub-gebieden kunnen bijdragen tot kankertherapie weerstand 13,14. Daarom is de mogelijkheid om cellen te analyseren in deze gebieden afzonderlijk is cruciaal inzicht tumorgeneesmiddel responsen.
De fluorescentie ubiquitinatie celcyclus indicator (Fucci) is gebaseerd op de rode (Kusabira Orange – KO) en groene (Azami Green – AG) fluorescerende tagging van CDT1 en geminin, die worden afgebroken in verschillende fasen van de celcyclus 15. Zo celkernen verschijnen rood in G1, geel in de vroege S en groen in S / G2 / M-fase. We beschrijven hier twee complementaire methoden beide met Fucci de celcyclus identificeren, samen met het gebruik van beeldverwerkingssoftware of kleurstofdiffusie flowcytometrie test om te bepalen of cellen in de G1 aangehouden midden of buitenste proliferatin verblijfG ring, en de afstand van de individuele cellen van de rand van de sferoïde. Deze methoden werden ontwikkeld in onze eerdere publicatie waarin we aangetoond dat melanoomcellen in hypoxische gebieden in het midden van het bolvormige of / en in aanwezigheid van gerichte therapieën kunnen in G1 arrest voor langere tijd blijft en opnieuw kan voer de celcyclus bij gunstiger voorwaarden ontstaan 7.
Protocol
Representative Results
Discussion
Semi-automatische beeldanalyse geïdentificeerd de bolvormige innerlijke G1 gearresteerd regio en uitdijende buitenste lagen. Deze werkwijze kan worden toegepast voor levende sferoïden behulp van een optische sectie of in vaste bolvormige gedeelten, veranderingen in niet alleen de celcyclus maar merkerexpressie (via immunofluorescentie), celdood of celmorfologie in de verschillende regio's te identificeren. Cel beweeglijkheid binnen de verschillende bolvormige regio kan ook worden gekwantificeerd – als live-confoca…
Declarações
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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