Imaging- 및 유동 세포 계측법 기반의 셀 위치의 분석 및 3D 흑색 종 회전 타원체의 세포주기
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
다세포 차원 타원체는 그러나 그들이 많은 고형암에 대한 체외 모델로 일반적인 사용에 온 것이 최근이다, 수십 년 동안 종양 모델로 알려져있다. 그들은 점점 복잡, 고가이며 시간 소모적 인 비보 모델에서와 간단한 저비용 차원 단층 모델 1-6 사이의 중간으로 높은 처리량 약물 발견 화면에 사용하고있다. 2D 문화 연구는 종종 생체 내에서 복제 할 수 없습니다. 많은 종류의 암의 회전 타원체 모델은 성장 특성, 약물 감수성, 약물 침투, 세포 – 세포 상호 작용, 고형 종양 6-11 생체 내에서 볼 산소 및 영양분과 괴사 현상의 제한된 유용성을 모방 할 수있다. 스페 로이드는 회전 타원체 (7)의 주변부 괴사 코어, 코어를 둘러싸는 대기 또는 G1 체포 영역 및 세포 증식을 개발한다. 이들 영역의 개발세포 밀도, 증식 속도 및 회전 타원체 (12)의 크기에 따라 달라질 수있다. 그러나, 이들 서로 다른 서브 – 지역에서 보이는 세포 이질성은 암 치료 용 저항 (13, 14)에 기여할 수 있음을 가정하고있다. 따라서, 이들 영역에서 세포를 분석하는 기능은 별도 이해 종양 약물 반응에 중요하다.
세포주기 (15)의 다른 단계에서 열화 Cdt1 및 geminin 형광 태그 – 녹색 (AG Azami 그린) – 형광 유비퀴틴 세포주기 표시기 (FUCCI) 시스템은 적색 (KO Kusabira 오렌지)에 기초한다. 따라서 세포 핵은 초 S 노란색, G1 빨간색과 S / G2 / M 단계에서 녹색 나타납니다. 우리는 여기에 두 개의 상보적인 방법을 기술 모두 셀 G1 체포 센터 혹은 외측 proliferatin에 상주하는지 여부를 결정하기 위해 이미징 소프트웨어 또는 분석 계측법 염료 확산 유동의 사용과 함께, 세포주기를 식별 FUCCI를 사용g 링과 회전 타원체의 에지로부터 각 셀의 거리. 또는 / 및 표적 치료의 존재하에 장시간 G1 체포에 남아있게하고, 재 수 이들 방법은 우리가 회전 타원체의 중심에 저산소 영역에서 그 흑색 종 세포를 보여 이전 공보, 개발되었다 보다 유리한 조건이 발생할 때 7 세포주기를 입력한다.
Protocol
Representative Results
Discussion
반 – 자동화 영상 분석은 내측 회전 타원체 G1 구속 영역을 식별하고, 외층 증식. 이 방법은 광학 부재를 이용하여 실시간 회전 타원체에 사용할 수도 있고, 고정 된 회전 타원체 부분에서, 서로 다른 영역에서의 세포주기하지만 (면역 통해) 마커의 발현, 세포 사멸 또는 세포 형태뿐만 아니라 변화를 식별하기. 다른 구형 영역 내의 세포 운동성은 정량화 될 수있다 – 셀 추적 이미지 분석 단계와 함?…
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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