Imaging- og flowcytometrisystemer basert analyse av Cell Plasser og cellesyklus i 3D melanom Spheroids
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
Flercellet 3D-kuler har vært kjent som et tumormodell i flere tiår, men det er bare nylig at de er kommet inn i mer vanlig bruk som en in vitro modell for mange faste cancere. De blir i økende grad brukt i high-throughput legemiddel skjermer som et mellomledd mellom komplisert, dyrt og tidkrevende in vivo-modeller og enkel, rimelig 2D enkeltlag modell 1-6. Studier i 2D kultur er ofte ikke i stand til å bli replikert in vivo. Sfæroide modeller av mange typer av cancer er i stand til å etterligne vekstegenskaper, medikamentsensitivitet, medikamentpenetrering, celle-celle-interaksjoner, begrenset tilgjengelighet av oksygen og næringsstoffer, og utvikling av nekrose som sees in vivo i faste tumorer 6-11. Sfæroider utvikle en nekrotisk kjerne, en stillestående eller G1 arrestert region som omgir kjernen, og prolifererende celler i periferien av den sfæroide 7. Utviklingen av disse regionenekan variere avhengig av celletetthet, proliferasjon hastighet og størrelsen av den sfæroide 12. Det er blitt antatt at den cellulære heterogenitet sett i disse forskjellige sub-regioner kan bidra til cancerterapi motstand 13,14. Derfor er evnen til å analysere cellene i disse regionene er separat avgjørende for forståelsen tumormedikamentresponser.
Fluorescensen ubiquitinering cellesyklus-indikator (FUCCI) Systemet er basert på den røde (Kusabira Orange – KO) og grønn (Azami Green – AG) fluorescerende merking av Cdt1 og geminin, som er nedbrutt i ulike faser av cellesyklusen 15. Dermed cellekjerner blir røde på G1, gult tidlig i S og grønt i S / G2 / M-fasen. Vi beskriver her to komplementære metoder både ved hjelp FUCCI for å identifisere cellesyklus, sammen med bruk av bilde programvare eller et fargestoff diffusjon strømningscytometri-analyse for å bestemme hvorvidt celler ligge i G1 arrestert senteret eller den ytre proliferating ringen, og avstanden av individuelle celler fra kanten av den sfæroide. Disse fremgangsmåter ble utviklet i vårt tidligere publikasjon, hvor Vi viste at melanomceller i hypoksiske områder i sentrum av den sfæroide eller / og i nærvær av målrettede terapier er i stand til å forbli i G1 arrest i lengre perioder, og kan re- gå inn i cellesyklusen når mer gunstige forhold oppstår 7.
Protocol
Representative Results
Discussion
Semi-automatisert bildeanalyse identifiserte spheroid indre G1 arrestert region, og voksende ytre lag. Denne fremgangsmåten kan brukes på levende sfæroider ved hjelp av en optisk del, eller i faste sfæroide seksjoner, for å identifisere endringer i ikke bare cellesyklusen, men da uttrykket (via immunofluorescens), celledød, eller cellemorfologi i disse forskjellige områder. Cellemotilitet innenfor ulike sfæroide områder kan også kvantifiseres – hvis levende konfokal tidsforløp avbildning sammen med en cellesp…
Disclosures
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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