Análisis basado en Citometría Imaging- y flujo de Cell posición y el ciclo celular en 3D melanoma esferoides
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
Esferoides multicelulares 3D se han conocido como un modelo de tumor durante décadas, sin embargo, es sólo recientemente que han llegado a ser de uso más común a medida un modelo in vitro para muchos cánceres sólidos. Ellos se utilizan cada vez en las pantallas de descubrimiento de fármacos de alto rendimiento como un intermediario entre el complejo, costoso y lento en modelos in vivo y el simple, el modelo de monocapa 2D bajo coste 6.1. Los estudios realizados en la cultura 2D a menudo no pueden ser replicados en vivo. Modelos Esferoide de muchos tipos de cáncer son capaces de imitar las características de crecimiento, sensibilidad a los fármacos, la penetración del fármaco, las interacciones célula-célula, la disponibilidad restringida de oxígeno y nutrientes y el desarrollo de la necrosis que se observa in vivo en tumores sólidos 6-11. Esferoides desarrollan un núcleo necrótico, una región de reposo detenido o G1 que rodea el núcleo, y las células que proliferan en la periferia del esferoide 7. El desarrollo de estas regionespuede variar dependiendo de la densidad celular, la tasa de proliferación y el tamaño del esferoide 12. Se ha planteado la hipótesis de que la heterogeneidad celular visto en estos diferentes sub-regiones puede contribuir a la resistencia a la terapia del cáncer 13,14. Por lo tanto la capacidad de analizar las células en estas regiones por separado es crucial para las respuestas de drogas comprensión tumorales.
El sistema indicador de ciclo celular ubiquitinación de fluorescencia (FUCCI) se basa en el rojo (Kusabira Orange – KO) y verde (verde Azami – AG) marcación fluorescente de Cdt1 y geminin, que se degrada en diferentes fases del ciclo celular 15. Así núcleos celulares aparecen rojos en G1, amarillo a principios de S y verde en S / G2 / M fase. Se describe aquí dos métodos complementarios tanto usando FUCCI para identificar el ciclo celular, junto con el uso de software de formación de imágenes o un flujo de difusión de colorante ensayo de citometría para determinar si las células residen en el centro G1 detenida o el exterior proliferating anillo, y la distancia de las células individuales desde el borde del esferoide. Estos métodos se desarrollaron en nuestra publicación anterior, en el que demostraron que las células de melanoma en regiones hipóxicas en el centro del esferoide o / y en presencia de terapias dirigidas son capaces de permanecer en arresto G1 durante largos períodos de tiempo, y pueden re- entrar en el ciclo celular cuando se presentan condiciones más favorables 7.
Protocol
Representative Results
Discussion
Análisis de imágenes semi-automatizado identifica la región G1 arrestado interior esferoide, y la proliferación de las capas exteriores. Este método puede ser utilizado en esferoides en vivo usando una sección óptica, o en secciones esferoide fijos, para identificar los cambios en no sólo el ciclo celular, pero la expresión del marcador (a través de inmunofluorescencia), la muerte celular, o la morfología celular en estas diferentes regiones. Motilidad celular dentro de las diferentes regiones esferoide tambi…
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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