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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		<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>
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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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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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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>
		</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>
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		<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>
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		<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>
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		<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>
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		<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>
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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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		<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>
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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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		<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>
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		<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>
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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. ...

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

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

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

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

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

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

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

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.

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

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

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

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 stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

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

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

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