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.
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.
Multicellulære sfæroider 3D har været kendt som en tumormodel i årtier, men det er først for nylig, at de er kommet ind i mere almindelige anvendelse som en in vitro model for mange faste cancere. De bliver i stigende grad brugt i high-throughput drug discovery-skærme som et mellemprodukt mellem komplekse, dyre og tidskrævende in vivo modeller og enkle, billige 2D monolag model 1-6. Studier i 2D kultur er ofte ude af stand til at blive gentaget in vivo. Sfæroide modeller af mange typer cancer er i stand til at efterligne egenskaberne vækst, medikamentfølsomhed lægemiddelpenetrering, celle-celle-interaktioner, begrænset tilgængelighed af ilt og næringsstoffer og udvikling af nekrose, der ses in vivo i faste tumorer 6-11. Sfæroider udvikle en nekrotisk kerne, en hvilende eller G1 standset område, der omgiver kernen, og prolifererende celler ved periferien af klumpformet 7. Udviklingen af disse regionerkan variere afhængigt af celletætheden, proliferationshastighed og størrelsen af sfæroide 12. Det er blevet antaget, at den cellulære heterogenitet ses i disse forskellige delregioner kan bidrage til cancerterapi resistens 13,14. Derfor evnen til at analysere celler i disse regioner særskilt er afgørende for forståelsen tumor narkotika reaktioner.
Systemet fluorescens ubiquitinering cellecyklus indikator (Fucci) er baseret på den røde (Kusabira Orange – KO) og grøn (Azami Green – AG) fluorescerende mærkning af Cdt1 og geminin, som nedbrydes i forskellige faser af cellecyklus 15. Således cellekerner ser røde i G1, gul i begyndelsen af S og grøn i S / G2 / M-fasen. Vi beskriver her to komplementære metoder både ved hjælp Fucci at identificere cellecyklus, sammen med anvendelse af imaging software eller et farvestof diffusion flowcytometriassayet at afgøre, om cellerne bor i G1 standset center eller den ydre proliferating ring, og afstanden af de enkelte celler fra kanten af sfæroide. Disse fremgangsmåder blev udviklet i vores tidligere publikation, hvor vi vist, at melanomceller i hypoxiske regioner i midten af sfæroid og / eller i nærværelse af målrettede behandlinger er i stand til at forblive i G1 arrest i længere perioder, og kan re- ind i cellens cyklus, når mere gunstige betingelser opstår 7.
Semi-automatiseret billedanalyse identificeret sfæroid indre G1 standset region og prolifererende ydre lag. Denne metode kan anvendes på levende sfæroider ved hjælp af en optisk sektion, eller i faste sfæroide sektioner, identificere forandringer i ikke kun cellecyklussen, men markørekspression (via immunfluorescens), celledød eller cellemorfologi i disse forskellige regioner. Cellemotilitet inden for forskellige sfæroide regioner kan også kvantificeres – hvis der tilsættes levende konfokal tidsforskydningen b…
The authors have nothing to disclose.
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).
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 |