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;

<table border="0" cellpadding="0" cellspacing="0" style="width:1292px;" width="1292">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="48">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="20">
			<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>
		</tr>
		<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>
		</tr>
		<tr height="40">
			<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="22">
			<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
		<tr height="25">
			<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>
		</tr>
	</tbody>
</table>

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. ...

The protocols presented here have been optimized for 3D culture and allow for efficient and affordable investigation of processes associated with cancer cell growth, proliferation, and death in a 3D environment. The main advantages of using 3D culture is that they are a unique possibility for recapitulating key aspects of the tumor microenvironment in vitro. 3D spheroid's an important tool for cancer drug screening, which is increasingly being used to improve translation between in vitro and in vivo conditions in cancer therapy research.Some of the protocols require a certain amount of practice and skill. This is in particular true for producing spheroids by the hand drop method, and for embedding spheroids for immunohistochemistry. As key elements of these procedures are difficult to describe in writing, such as how to insert spheroids into a drop of agarose, visual demonstration will help researchers perform these procedures.Dilute the prepared cell suspension in a 15 milliliter tube to obtain the optimal cell density. Transfer the diluted cell suspension to a sterile reservoir, and use a multi-channel pipette to dispense 200 microliters into the previously prepared plate. Incubate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity.Every two to three days, acquire light microscopic images of the spheroids. After acquiring the images, remove 100 microliters of the spent medium from each well, and replace it with 100 microliters of fresh medium. First, thaw reconstituted basement membrane on ice.Keep any individually-wrapped plates and reservoirs on ice before use. Next, dilute the prepared cell suspension in a 15-milliliter tube to obtain the optimal cell density. Place the tube containing the diluted cell suspension on ice.Fill the plastic containers with ice and transfer them into the hood. Transfer the chilled plates and reservoirs to a cell culture hood. Place the plates and reservoirs back on ice.Resuspend the rBM gently to ensure a homogenous gel. Add the optimal concentration of the rBM to the chilled cell suspensions. Invert the tube to ensure that the rBM and cell suspension are properly mixed.Then, transfer this mixture to a sterile reservoir, and use a multichannel pipette to dispense 200 microliters to each well of a chilled, ultra-low-attachment, 96-well plate. Centrifuge the plate at 750 times g for 15 minutes to ensure that the cells are clustered together when the rBM hardens. Use the centrifuge soft descent or minimal braking function.First, add six milliliters of PBS to a cell culture dish. Dilute the prepared cell suspension to obtain a suitable dilution. A practical dilution is 50, 000 cells per milliliter.Pour the cell suspension into a sterile reservoir and use a multichannel pipette to carefully place up to 30 drops of suspension into the lid of the cell culture dish, resulting in a concentration of 2000 cells per drop. In a quick but controlled movement, invert the lid and place it on top of the cell culture dish containing PBS. Incubate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity for four to six days.If the spheroids are to be used for protein lysates or embedding, pull them by removing the lid, tilting it, and then washing down the drops with one milliliter of heated media. Transfer the resulting spheroid-containing medium to a 1.5 milliliter tube. Let the spheroid settle to the bottom of the tube before proceeding with protein lysates or embedding.Cut the end of a P200 pipette tip to more easily capture the spheroids without disturbing their structure. For each condition, pull a minimum of 12 but ideally between 18 and 24 spheroids in a 1.5 milliliter tube. Place the tubes on ice and allow the spheroids to settle at the bottom.Next, move from the sterile cell laboratory to the regular laboratory. Wash the spheroids twice in one milliliter of ice-cold one X PBS, making sure to let the spheroids settle before removing the PBS between each washing step. Then, aspirate as much PBS as possible without disturbing or removing the spheroids.Add five microliters of heated lysis buffer with phosphatase and protease inhibitors per spheroid. Vortex the spheroids for 30 seconds and then perform a quick centrifugation for 10 seconds. Repeat these intervals of vortexing and centrifugation for five to 10 minutes, or until the spheroids are dissolved.After preparing the PI solution, remove 100 microliters of medium from each well in the 96-well plate, making sure to not remove the spheroids. Add 100 microliters of heated one X PBS to each well, followed by removing 100 microliters of liquid from the wells. Repeat this washing step three times to wash out any remaining medium.Next, add 100 microliters of the PI solution to each well. Cover the plate in aluminum foil and incubate for 37 degrees Celsius with 5%carbon dioxide and 95%humidity for 10 to 15 minutes. After this, repeat the washing process with heated one X PBS three times, as previously described, to wash out the PI solution and diminish the background signal when imaging.Using an epifluorescence microscope, image the spheroids. Within the imaging software, open the multi-channel menu. Set the exposure time for the relevant channels, and press Read Settings after adjusting each channel.Open the Z-stack menu and define the start and end by adjusting the image focus. Initially have the spheroid out of focus, then move the focal point through the entire spheroid until the image becomes blurry once again. Then adjust the step size to be between 18 to 35 micrometers, and press Start.To begin, fix the spheroids in a fume hood on day one as outlined in the text protocol. On day two, place the agarose gel into a water-filled beaker in a microwave oven to heat it and keep the gel warm on a bench-top heating plate set to 60 degrees Celsius, until needed. Wash the spheroids twice in the fume hood using one milliliter of ice-cold one X PBS per wash.Then, aspirate most of the PBS. Prepare a 20 microliter pipette tip by cutting it at an incline to obtain a pointier tip with a larger hole. After this, make an agarose gel drop on a microscope slide.Place the slide on a warm heating block to prevent the agarose from solidifying. Using the modified pipette tip, catch as many spheroids as possible in a volume of 15 to 20 microliters. Carefully inject the spheroids into the center of the agarose gel drop, making sure to not touch the microscope slide.Incubate at room temperature or at four degrees for five to 10 minutes to let the agarose gel drop harden. When the gel drop has solidified somewhat, but is still rather soft, use a scalpel to carefully push it from the microscope slide into a plastic tissue cassette. Transfer the tissue cassette to a beaker filled with ethanol and store at room temperature.In this study, a series of methods are presented for the analysis of anti-cancer treatment-induced changes in cancer cell viability and death in 3D culture. The concentration of rBM added can profoundly affect the morphology of the spheroids. The addition of up to 2.5%rBM allows spheroid formation in SKBr-3 breast cancer cells, with no further effect at higher concentrations.In contrast, BxPC-3 pancreatic cancer cells spontaneously form small compact spheroids. In this cell type, increasing the rBM to 1.5%or above elicits a distinct morphological change from spheroid to more convoluted structures with protrusions and invaginations. An example of the use of spheroid cultures for screening of chemotherapy efficacy is shown here.Light microscope images are acquired every two to three days during a dose response experiment performed to determine the dose necessary for 50%reduced viability in MDA-MB-231 breast cancer cells. The spatial arrangement of dead cells upon an increasing concentration of an inhibitor can be visualized using PI staining. As seen, control spheroids show a limited necrotic, late apoptotic core, whereas the dead cells are distributed throughout the spheroid as the concentration of inhibitor is increased.The spheroid technique shown here can be applied to many other assays, such as 3D invasion, migration, or microphoretic analysis. It is also applicable for co-cultures with fibroblast, adipocytes, or immune cells, which can provide important information about cellular interactions in the tumor microenvironment. The development of this technique has been very important in elucidating the role of the tumor microenvironment in numerous aspects of cancer development, including gene expression, invasion, and treatment assistance.

assessing-cell-viability-death-3d-spheroid-cultures-cancer

Research

JoVE Journal

Cancer Research

26.1K Views.  University of Copenhagen. The protocols presented here have been optimized for 3D culture and allow for efficient and affordable investigation of processes associated with cancer cell growth, proliferation, and death in a 3D environment. The main advantages of using 3D culture is that they are a unique possibility for recapitulating key aspects of the tumor microenvironment in vitro. 3D spheroid's an important tool for cancer drug screening, which is increasingly being used to improve translation between in vitro and ...