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).

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PerkinElmer contributed to the costs of publication.

半自動画像解析は、回転楕円体の内側G1逮捕領域を同定し、そして外層の増殖。この方法は、これらの異なる領域で（免疫蛍光により）、細胞周期が、マーカー発現しないだけの変化を識別するために、光学部を使用して、ライブスフェロイドに、または一定の回転楕円体のセクションで使用され、細胞死、または細胞の形態であってもよいです。異なる回転楕円体領域内の細胞の運動性も?...

多3Dスフェロイドが数十年のための腫瘍モデルとして知られている、しかし、それは彼らが多くの固形癌のためのin vitroモデルとして、より一般的に使用されるようになったことはごく最近です。彼らはますます複雑、高価で時間のかかる生体内モデルで 、シンプルで低コストの2D単層モデル1-6との間の中間のような高スループットの薬物発見スクリーニングにおいて使用されています。 2D培養における研究は、多くの場合、 生体内で複製することができません。癌の多くの種類の回転楕円体モデルは、増殖特性、薬剤感受性、薬剤の浸透、細胞-細胞相互作用、固形腫瘍6-11 においてインビボで見られる酸素や栄養素および壊死の開発の制限された利用可能性を模倣することができます。スフェロイドは、回転楕円体7の周囲に壊死性コア、コアを取り囲む逮捕静止またはG1領域、および増殖細胞を開発しています。これらの領域の開発細胞密度、増殖速度および回転楕円体12の大きさに応じて変えることができます。これらの異なるサブ領域で見られる細胞の異質性は、癌治療抵抗13,14に寄与し得ると仮定されています。したがって、これらの領域内の細胞を分析する能力は、個別の腫瘍薬物応答を理解するために重要です。 緑（あざみグリーン- AG）は、細胞周期15の異なる段階に分解されるCDT1とGemininの蛍光タグ付け、 -蛍光ユビキチン化、細胞周期のインジケータ（FUCCI）システムは、赤（KO Kusabiraオレンジ）に基づいています。従って、細胞核は、初期のS内の黄色、G1の赤とS / G2 / M期で緑に表示されます。ここでは二つの相補的な方法を記載している両方の細胞がG1逮捕中央または外側proliferatinに存在するかどうかを決定するために画像処理ソフトウェアまたは染料拡散フローサイトメトリーアッセイの使用に伴って、細胞周期を特定するためにFUCCIを用いグラムリング、及び回転楕円体の端部から個々の細胞の距離。これらの方法は、我々は回転楕円体の中心にあるおよび/または標的療法の存在下での低酸素領域におけるメラノーマ細胞が長期間G1停止のままにすることが可能であり、再できることを実証した我々の以前の出版物、に開発されましたより有利な条件は7を生じたとき、細胞周期を入力してください。

<table><tbody><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>agarose low melting point</td><td>Life Technologies</td><td>16520-050</td><td>For sectioning</td></tr><tr><td>noble agar&amp;nbsp;</td><td>Sigma</td><td>A5431</td><td>For making spheroids</td></tr><tr><td>agarose for spheroids</td><td>Fisher Scientific</td><td>BP1356-100</td><td>For making spheroids</td></tr><tr><td>0.05% trypsin/EDTA</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>14175-103</td><td></td></tr><tr><td>10% formalin</td><td>Sigma</td><td>HT5014-1CS</td><td>CAUTION: Harmful, corrosive. Use Personal Protective Equipment, do &amp;nbsp;not breath fumes (open in a fume cupboard).</td></tr><tr><td>live/dead near IR</td><td>Life Technologies</td><td>L10119</td><td></td></tr><tr><td>vibratome</td><td>Technical Products International, Inc</td><td></td><td></td></tr><tr><td>coulture cup</td><td>Thermo-Fisher Scientific</td><td>SIE936</td><td>Mold for sectioning spheroids</td></tr><tr><td>hemocytometer</td><td>Sigma</td><td>Z359629</td><td></td></tr><tr><td>96-well tissue culture plate</td><td>Invitro</td><td>FAL353072</td><td></td></tr><tr><td>collagenase</td><td>Sigma</td><td>C5138&amp;nbsp;</td><td></td></tr><tr><td>confocal microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Flow cytometer analyser</td><td>Becton Dickinson</td><td>LSRFortessa</td><td></td></tr><tr><td>volocity</td><td>PerkinElmer</td><td></td><td>Imaging software</td></tr><tr><td>flowjo</td><td>Tree Star</td><td></td><td>Flow cytometry software</td></tr><tr><td>Vaccuum grease</td><td>Sigma</td><td>Z273554</td><td></td></tr><tr><td>Mounting media</td><td>Vector Laboratories</td><td>H1000</td><td></td></tr><tr><td>FUCCI (commercial constructs)</td><td>Life Technologies</td><td>P36238</td><td>Transient transfection only</td></tr><tr><td>Cell strainer 70 &amp;#956;m</td><td>In Vitro</td><td>FAL352350</td><td></td></tr><tr><td>Round bottom 5 ml tubes (sterile)</td><td>In Vitro</td><td>FAL352003</td><td></td></tr><tr><td>Round bottom 5 ml tubes (non-sterile)</td><td>In Vitro</td><td>FAL352008</td><td></td></tr></tbody></table>

imaging- and flow cytometry-based analysis of cell position and the cell cycle in 3d melanoma 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.

腫瘍スフェロイドを生成するいくつかの方法は、このプロトコルは、細胞は、寒天またはアガロース3,7,9上で培養された非接着性成長法を使用して、存在する。 図1は、寒天上の3日後、C8161メラノーマ回転楕円体の一例を示す図です。 C8161スフェロイドは500の直径との定期的なサイズのスフェロイド形成 - 600μmで3日後（= 565、SD = 19、n = 3の平均値）。スフェロイドを形成?...

Watch this Scientific Journal Video about Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids at JoVE.com

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

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.

Imaging-とフローサイトメトリーベースのセル位置の解析と3Dメラノーマスフェロイドにおける細胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

imaging-flow-cytometry-based-analysis-cell-position-cell-cycle-3d

Research

JoVE Journal

Medicine

13.0K ビュー.  The Centenary Institute. 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. 

ビデオ: Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

黒色腫切片 3 D 回転楕円体の皮膚モデル

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

がん研究

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

光コヒーレンストモグラフィーを用いた三次元腫瘍回転楕円体の縦断的形態学的および生理学的監視

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

がん幹細胞を標的とする抗腫瘍性薬物スクリーニングのための生理学的患者由来3Dスフェロイド

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

癌細胞の3Dスフェロイド培養における細胞生存率と死の評価

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

がん関連線維芽細胞分化とインビトロ研究における侵入のためのより生理学的システムとしての3Dスフェロイドモデル

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

腫瘍間質相互作用と創薬を研究するための新規ストロマ線維芽細胞変調3D腫瘍球状モデル

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the spheroid at the cellular level is important for analysis, yet existing techniques are often expensive, laborious, use non-standard equipment, cause significant size distortion, or are limited to relatively small spheroids. This protocol presents a new method of mounting and clearing spheroids that address these issues while allowing for confocal analysis of the inner structure of spheroids. In contrast to existing approaches, this protocol provides for rapid mounting and clearing of a large number of spheroids using standard equipment and laboratory supplies. Mounting spheroids in a pH-neutral agarose-PBS gel solution before introducing a refractive-index-matched clearing solution minimizes size distortion common to other similar techniques. This allows for detailed quantitative and statistical analysis where the accuracy of size measurements is paramount. Furthermore, compared to liquid clearing solutions, the agarose gel technique keeps spheroids fixed in place, allowing for the collection of three-dimensional (3D) confocal images. The present article elaborates how the method yields high-quality two- and 3D images that provide information about inter-cell variability and inner spheroid structure.

Tumor spheroids are becoming increasingly utilized to assess tumor cell-microenvironment interactions and therapy response. The present protocol describes a robust but simple method for semi-high-throughput imaging of 3D tumor spheroids using rapid optical clearing.

腫瘍スフェロイドの半ハイスループット分析のための迅速な光学的クリアリング

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三次元（3D）腫瘍スフェロイド浸潤アッセイ

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

医学

<meta charset="utf8"></head><body>3Dコラーゲンマトリックスセットアップにおけるマクロファージ-腫瘍スフェロイド相互作用の検討

A Macrophage-Tumor Spheroid Co-Invasion Assay

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few protocols are available that allow an in vitro approximation of the in vivo situation. Here, we present a novel approach to observing the impact of human macrophages on the invasiveness of cancer cells, using tumor spheroids of H1299 non-small cell lung carcinoma cells embedded in a three-dimensional (3D) collagen I matrix. With this co-cultivation setup, we tested the impact of small interfering RNA (siRNA)-based depletion of regulatory factors in macrophages on the 3D invasion of cancer cells from the tumor spheroid compared to controls. This method allows the determination of different parameters, such as spheroid area or the number of invading cancer cells, and thus, to detect differences in cancer cell invasion. In this article, we present the respective setup, discuss the subsequent analysis, as well as the advantages and potential pitfalls of this method.

<meta charset="utf8"></head><body>マクロファージ-腫瘍スフェロイド共浸潤アッセイ

Here, we present a protocol to investigate the interaction of primary human monocyte-derived macrophages with a tumor spheroid in a three-dimensional (3D) collagen I matrix, with the possibility to compare the impact of soluble and physical properties of the microenvironment on cell invasion.

マクロファージ-腫瘍スフェロイド共浸潤アッセイ

The interaction of immune and cancer cells and their respective impact on metastasis represents an important aspect of cancer research. So far, only a few ...

Imaging-とフローサイトメトリーベースのセル位置の解析と3Dメラノーマスフェロイドにおける細胞周期

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