Imaging- och flödescytometri-baserad analys av cell Position och cellcykeln i 3D Melanom Sfäroider
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
Flercellig 3D sfäroider har varit känd som en tumörmodell i årtionden, men det är först nyligen som de har kommit in i mer allmänt bruk som en förebild för många fasta cancer in vitro. De används alltmer i hög genomströmning läkemedelsforskning skärmar som en intermediär mellan komplexa, dyra och tidsödande in vivo-modeller och enkel, billig 2D monolager modell 1-6. Studier i 2D kultur är ofta inte kan replikeras in vivo. Sfäroid modeller av många typer av cancer kan efterlikna egenskaper tillväxt, läkemedelskänslighet, läkemedelspenetrations, cell-cell interaktioner, begränsad tillgänglighet av syre och näringsämnen och utveckling av nekros som ses in vivo i solida tumörer 6-11. Sfäroider utvecklar en nekrotisk kärna, en vilande eller G1 arrested område som omger kärnan, och prolifererande celler vid periferin av den sfäroid 7. Utvecklingen av dessa regionerkan variera beroende på celldensiteten, proliferationshastighet och storleken på den sfäroid 12. Det har antagits att den cellulära heterogeniteten ses i dessa olika delregionerna kan bidra till cancerterapi motstånd 13,14. Därför förmåga att analysera celler i dessa regioner är separat avgörande att förstå tumörläkemedelssvar.
Fluorescens ubikvitinering cellcykelindikator (Fucci) Systemet bygger på den röda (Kusabira Orange – KO) och grön (Azami Green – AG) fluorescerande märkning av Cdt1 och geminin, som bryts ned i olika faser av cellcykeln 15. Således cellkärnor visas rött i G1, gul i början av S och grön i S / G2 / M-fasen. Vi beskriver här två komplementära metoder både med användning Fucci att identifiera cellcykeln, tillsammans med användning av bildgivande programvara eller en färgdiffusion flödescytometri analys för att bestämma huruvida celler är bosatta i G1 arrested centrum eller det yttre proliferating ring, och avståndet av enskilda celler från kanten av sfäroid. Dessa metoder har utvecklats i vår tidigare offentliggörande, där vi visat att melanomceller i hypoxiska regioner i mitten av sfäroid och / eller i närvaro av riktade behandlingar kan vara kvar i G1 gripande under längre tidsperioder, och kan åter ange cellcykeln när mer gynnsamma förhållanden uppstår 7.
Protocol
Representative Results
Discussion
Halvautomatisk bildanalys identifierade sfäroid inre G1 greps regionen, och prolifererande yttre skikt. Denna metod kan användas på levande sfäroider med hjälp av en optisk avsnitt, eller i fasta sfäroida sektioner, för att identifiera förändringar i inte bara cellcykeln men markör uttryck (via immunofluorescens), celldöd, eller cellmorfologin i dessa olika områden. Cellmotiliteten inom olika sfäroida regioner kan också kvantifieras – om levande konfokal time lapse avbildning tillsammans med en spårning c…
Divulgazioni
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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