Name: assessing cell viability and death in 3d spheroid cultures of cancer cells
Uploaded: 2019-06-16T14:00:00
Description: Watch this Scientific Journal Video about Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells at JoVE.com

We are grateful to Katrine Franklin Mark and Annette Bartels for excellent technical assistance and to Asbj&#248;rn N&#248;hr-Nielsen for performing the experiments in Figure 1D. This work was funded by the Einar Willumsen Foundation, the Novo Nordisk Foundation, and Fondation Juchum (all to SFP).

1. Generation of Spheroids
<ol>
	<li>Preparing cell suspensions for spheroid formation 
	NOTE: Different cell lines have very different adhesion properties and the most suitable spheroid formation protocol must be established in each case. We have found&#160;that MCF-7 and BxPC-3 cells are suitable for spontaneous spheroid formation, while MDA-MB-231, SKBr-3, Panc-1 and MiaPaCa require the addition of reconstituted basement membrane to successfully form spheroids. Only MDA-MB-231 and BxPC-3 cells ...

The use of 3D cancer cell spheroids has proven a valuable and versatile tool not only for anticancer drug screening, but also for gaining mechanistic insight into the regulation of cancer cell death and viability under conditions mimicking those in the tumor microenvironment. This is particularly crucial as the accessibility, cellular uptake, and intracellular effects of chemotherapeutic drugs are profoundly impacted by the physico-chemical conditions in the tumor, including pH, oxygen tension, tortuosity, and physical a...

The use of multicellular spheroid models in cancer biology is several decades old1,2, but has gained substantial momentum in recent years. In large part, this reflects increased awareness of how strongly the phenotype of cancer cells is dependent on their microenvironment and specific growth conditions. The microenvironment in solid tumors is fundamentally different from that in corresponding normal tissues. This includes physico-chemical conditions such as pH, oxygen tension, as well as interstitial pressure, concentration gradients of soluble factors such as nutrients, waste products, and secreted signaling compounds (growth factors, cytokines). Furthermore, it includes the organization of the extracellular matrix (ECM), cell-cell interactions and intercellular signaling, and other aspects of the particular three-dimensional (3D) architecture of the tumor3,4,5,6. The specific microenvironmental conditions in which cancer cells exist, profoundly affect their gene expression profile and functional properties, and it is clear that, compared to that of cells grown in 2D, the phenotype of 3D spheroids much more closely mimics that of in vivo tumors7,8,9,10,11. 2D models, even if they employ hypoxia, acidic pH, and high lactate concentrations to mimic known aspects of the tumor microenvironment, still fail to capture the gradients of physico-chemical parameters arising within tumors, as well as their 3D tumor architecture. On the other hand, animal models are costly, slow, and ethically problematic, and generally, also have shortcomings in their ability to recapitulate human tumor conditions. Consequently, 3D spheroids have been applied as an intermediate complexity model in studies of a wide range of properties of most solid cancers9,11,12,13,14,15,16,17.
A widely employed use of 3D spheroids is in screening assays of anticancer therapy efficacy9,18,19,20. Treatment responses are particularly sensitive to the tumor microenvironment, reflecting both the impact of the tortuosity, restricted diffusion, high interstitial pressure, and acidic environmental pH on drug delivery, and the impact of hypoxia and other aspects of the microenvironment on the cell death response9,17. Because the environment within 3D spheroids inherently develops all of these properties7,8,9,10,11, employing 3D cell cultures can substantially improve the translation of results to in vivo conditions, yet allow efficient and affordable high-throughput screening of the net growth. However, the great majority of studies on the drug response of cancer cells are still carried out under 2D conditions. This likely reflects that, while some assays can relatively easily be implemented for 3D cell cultures, many, such as viability assays, western blotting, and immunofluorescence analysis, are much more conveniently done in 2D than in 3D.
The aim of the present work is to provide easily amenable assays and precise protocols for analyses of the effect of treatment with anti-cancer drugs on cancer cell viability and survival in a 3D tumor mimicking setting. Specifically, we provide and compare three different methods for spheroid formation, followed by methods for qualitative and quantitative analyses of growth, viability and drug response.&#160;

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			<td height="20" style="height:20px;width:408px;">2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI)</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:264px;"># C10595&#160;</td>
			<td style="width:336px;">For staining nuclei</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:408px;">5-Fluorouracil (5-FU)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:264px;">#F6627</td>
			<td style="width:336px;">Component in chemotherapeutic treatment</td>
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			<td height="22" style="height:22px;width:408px;">5-(N-ethyl-isopropyl) amiloride (EIPA)</td>
			<td style="width:284px;">Life Technologies</td>
			<td style="width:264px;">#E3111</td>
			<td style="width:336px;">Inhibitor of NHE1</td>
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			<td height="22" style="height:22px;width:408px;">Antibody against PARP and cPARP</td>
			<td style="width:284px;">Cell signaling</td>
			<td>#9542</td>
			<td style="width:336px;">Used in western blotting</td>
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			<td height="22" style="height:22px;width:408px;">Antibody against Ki-67</td>
			<td style="width:284px;">Cell signaling</td>
			<td style="width:264px;">#9449</td>
			<td style="width:336px;">Used for IHC</td>
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			<td height="22" style="height:22px;width:408px;">Antibody against p53</td>
			<td style="width:284px;">Cell Signaling&#160;</td>
			<td style="width:264px;">#2524&#160;</td>
			<td style="width:336px;">Used for IHC</td>
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			<td height="22" style="height:22px;width:408px;">Antibody against &#946;-actin</td>
			<td style="width:284px;">Sigma&#160;</td>
			<td style="width:264px;">A5441</td>
			<td style="width:336px;">Used in western blotting</td>
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			<td height="22" style="height:22px;width:408px;">Bactoagar</td>
			<td style="width:284px;">BD Bioscience</td>
			<td style="width:264px;">#214010</td>
			<td style="width:336px;">Used for agarose gel preparation</td>
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			<td height="22" style="height:22px;width:408px;">Benchmark protein ladder</td>
			<td style="width:284px;">Invitrogen</td>
			<td>#10747-012</td>
			<td style="width:336px;">Used for SDS-PAGE</td>
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			<td height="22" style="height:22px;">Bio-Rad DC Protein Assay kit</td>
			<td>Bio-Rad Laboratories</td>
			<td>#500-0113, #500-0114, #500-0115&#160;&#160;</td>
			<td style="width:336px;">Used for protein determination from lysates</td>
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			<td height="21" style="height:21px;width:408px;">B&#252;rker chamber</td>
			<td style="width:284px;">Marienfeld</td>
			<td style="width:264px;">610311</td>
			<td style="width:336px;">For cell counting&#160;</td>
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			<td height="21" style="height:21px;width:408px;">BX63 epifluoresence microscope</td>
			<td style="width:284px;">Olympus</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Used for fluorescent imaging</td>
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			<td height="21" style="height:21px;width:408px;">CellTiter-Glo 3D Cell Viability Assay</td>
			<td style="width:284px;">Promega</td>
			<td style="width:264px;">#G9681</td>
			<td style="width:336px;">Used for the cell viability assay</td>
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			<td height="21" style="height:21px;width:408px;">Cisplatin</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:264px;">#P4394&#160;</td>
			<td style="width:336px;">Component in chemotherapeutic treatment</td>
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			<td height="48" style="height:48px;width:408px;">Corning Spheroid Microplate, 96 well, Black with clear round bottom,&#160; Ultra-low attachment, With lid, Sterile</td>
			<td style="width:284px;">Corning</td>
			<td style="width:264px;">#4520</td>
			<td style="width:336px;">Used for growing spheroids with luminescence measurements as end point</td>
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			<td height="42" style="height:43px;width:408px;">Corning 96 well, clear round bottom,&#160; Ultra-low attachment microplate, With lid, Sterile</td>
			<td style="width:284px;">Corning</td>
			<td style="width:264px;">#7007</td>
			<td style="width:336px;">Sufficient for spheroid growth without luminescence measurements as end point</td>
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			<td height="22" style="height:23px;">Criterion TGX Precast Gels</td>
			<td style="width:284px;">Bio-Rad</td>
			<td style="width:264px;">5671025</td>
			<td style="width:336px;">Used for SDS-PAGE</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Doxorubicin</td>
			<td style="width:284px;">Abcam</td>
			<td style="width:264px;">#120629</td>
			<td style="width:336px;">Component in chemotherapeutic treatment</td>
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			<td height="20" style="height:20px;width:408px;">FLUOStar Optima Microplate reader</td>
			<td style="width:284px;">BMG Labtech</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Used for recording luminescence&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Formaldehyde&#160;</td>
			<td style="width:284px;">VWR Chemicals&#160;</td>
			<td style="width:264px;">#9713.1000&#160;</td>
			<td style="width:336px;">Used for cell fixation</td>
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			<td height="40" style="height:40px;width:408px;">Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td style="width:284px;">Gibco</td>
			<td style="width:264px;">#A1413202</td>
			<td style="width:336px;">Keep at 4 &#176;C to prevent solidification. Referred to as rBM in the protocol.</td>
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			<td height="25" style="height:25px;width:408px;">Heat-inactivated FBS</td>
			<td>Sigma</td>
			<td style="width:264px;">#F9665</td>
			<td style="width:336px;">Serum for growth media</td>
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			<td height="22" style="height:23px;width:408px;">ImageJ</td>
			<td style="width:284px;">NIH</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Scientific Image analysis</td>
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			<td height="22" style="height:22px;">Medim Uni-safe casette</td>
			<td>Medim Histotechnologie</td>
			<td>10-0114</td>
			<td style="width:336px;">Used for storage of embedded spheroids</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Mini protease inhibitor cocktail tablets</td>
			<td style="width:284px;">Roche Diagnostics GmBH&#160;</td>
			<td style="width:264px;"># 11836153001</td>
			<td style="width:336px;">Used for lysis buffer preparation</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:408px;">MZ16 microscope</td>
			<td style="width:284px;">Leica</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Used for light microscopic images</td>
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		<tr height="22">
			<td height="22" style="height:22px;">NuPAGE LDS 4x Sample Buffer&#160;</td>
			<td>Invitrogen</td>
			<td>#NP0007</td>
			<td style="width:336px;">Used for western blotting</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Pierce ECL Western blotting substrate</td>
			<td style="width:284px;">Thermo scientific</td>
			<td>#32106</td>
			<td>Used for western blotting</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Ponceau S</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>#P7170-1L</td>
			<td style="width:336px;">Used for protein band staining</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Prism 6.0</td>
			<td style="width:284px;">Graphpad</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Scientific graphing and statistical software</td>
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			<td height="25" style="height:25px;width:408px;">Propidium iodide (1mg/ml solution in water)</td>
			<td>Invitrogen&#160;</td>
			<td>P3566</td>
			<td>Light sensitive&#160;</td>
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			<td height="22" style="height:22px;width:408px;">Sterile reservoirs, multichannel</td>
			<td style="width:284px;">SPL lifesciences</td>
			<td style="width:264px;">21002</td>
			<td style="width:336px;">Used for seeding cells for spheroid formation</td>
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			<td height="25" style="height:25px;width:408px;">Superfrost Ultra-Plus Adhesion slide&#160;</td>
			<td style="width:284px;">Menzel-Gl&#228;ser</td>
			<td style="width:264px;">#J3800AMNZ</td>
			<td style="width:336px;">Microscope glass slide used for embedding</td>
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			<td height="25" style="height:25px;width:408px;">Tamoxifen</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:264px;">#T5648</td>
			<td style="width:336px;">Used as chemotherapeutic treatment</td>
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			<td height="25" style="height:25px;">Trans-blot Turbo 0.2 &#181;m nitrocellulose membranes</td>
			<td style="width:284px;">Bio-Rad</td>
			<td>#170-4159</td>
			<td style="width:336px;">Used for western blotting</td>
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			<td height="25" style="height:25px;width:408px;">Tris/Glycine/SDS running buffer&#160;</td>
			<td style="width:284px;">Bio-Rad&#160;</td>
			<td>#161 0732</td>
			<td style="width:336px;">Used for SDS-PAGE</td>
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			<td height="25" style="height:25px;width:408px;">Trypsin-EDTA solution</td>
			<td style="width:284px;">Sigma</td>
			<td>#T4174&#160;</td>
			<td style="width:336px;">Cell dissociation enzyme</td>
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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.

Spheroid growth assays based on the spheroid formation protocol schematically illustrated in Figure 1A and Figure 1B, were used as a starting point for analysis of the effects of anti-cancer drug treatments in a 3D tumor mimicking setting. The ease with which spheroids are formed is cell line specific, and some cell lines require supplementation with rBM in order to form coherent spheroids22. The concentr...

Watch this Scientific Journal Video about Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells at JoVE.com

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

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.

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

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

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Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

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Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

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Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

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Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

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Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...