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 have been employed for the hanging drop protocol, however other cell lines are certainly applicable.
	<ol>
		<li>Grow cells as monolayer until 70-80% confluency.</li>
		<li>Wash cells with phosphate buffered saline (1x PBS, 5 mL for a 25 cm2 or 10 mL for a 75 cm2 flask), add the cell dissociation enzyme (0.5 mL for a 25 cm2 or 1 mL for a 75 cm2 flask) and incubate the cells for 2-5 min at 37 &#176;C in 5% CO2 and 95% humidity.</li>
		<li>Check the cell detachment under a microscope and neutralize the cell dissociation enzyme by adding growth medium (6-10% serum depending on the cell line) to a total volume of 5 mL in a 25 cm2 or 10 mL for a 75 cm2 flask.</li>
		<li>Use a B&#252;rker chamber to count cells and count 8 squares in the chamber per cell preparation to obtain a high reproducibility of the size of the spheroids. 
		NOTE: Three protocols each describing a different method for spheroid formation are presented below. Protocol 1.2 and 1.3 can be used for all the subsequent analytic protocols presented, whereas protocol 1.4 is best suited for embedding and lysate preparations. Depending on the cell line, spheroid formation takes 2-4 days, irrespective of the method used.</li>
	</ol>
	</li>
	<li>Spontaneous spheroid formation
	<ol>
		<li>Perform steps 1.1.1-1.1.4.</li>
		<li>Dilute the cell suspension in a 15 mL tube to obtain 0.5-2 x 104 cells/mL (optimal cell density needs to be determined for each cell line) (Figure 1A (ii)).</li>
		<li>Fill the outer ring of wells with 1x PBS or growth medium to reduce evaporation from the remaining wells.&#160;Transfer the cell suspension to a sterile reservoir and, using a multichannel pipette, dispense 200 &#181;L/well into ultra-low attachment 96-well round bottom plates (Figure 1A (iii)).</li>
		<li>Incubate the plate in an incubator at 37 &#176;C with 5% CO2, 95% humidity.</li>
		<li>Every 2-3 days acquire light microscopic images of the spheroids. 
		NOTE: The images in this paper are taken at 11.5x magnification, which is appropriate for most spheroids prepared using these protocols.</li>
		<li>Every 2-3 days (after acquiring images) replace 100 &#181;L of medium (remove 100 &#181;L of the spent medium and replace with 100 &#181;L of fresh medium. 
		NOTE: To avoid removing spheroids when replacing medium, it is advisable to tilt the plate a bit while slowly removing the medium and inspect the aspirated medium in the tips for visible spheroids before discarding it.</li>
	</ol>
	</li>
	<li>Reconstituted basement membrane-mediated spheroid formation. 
	NOTE: Lactose dehydrogenase elevating virus (LDEV)-free reduced growth factor reconstituted basement membrane (rBM) was used. rBM is temperature-sensitive and should always be kept on ice, as it will clot if it reaches 15 &#176;C. Thaw the rBM on ice either overnight at 4 &#176;C or 2-4 h at room temperature (RT) before plating.
	<ol>
		<li>Thaw rBM on ice (see Table of Materials).</li>
		<li>Keep plates and reservoirs (if individually wrapped) on ice before use.</li>
		<li>Perform steps 1.1.1-1.1.4.</li>
		<li>Fill the outer ring of wells with 1x PBS or growth medium to reduce evaporation from the remaining wells. Dilute the cell suspension in a 15 mL tube to obtain 0.5-2 x 104 cells/mL (optimal cell density needs to be determined for each cell line) (Figure 1A (ii)).</li>
		<li>Place the 15 mL tube containing the diluted cell suspension on ice (e.g., in glass beaker) (Figure 1A (iia)).</li>
		<li>Transfer the chilled plates and reservoirs to the hood. Rinse plastic containers, fill them with ice and transfer them into the hood to allow the plates and reservoirs to be placed on ice during the entire procedure.</li>
		<li>Resuspend rBM gently to ensure a homogenous gel.</li>
		<li>Add 1-2% rBM (optimal concentration needs to be determined for each cell line) to the chilled cell suspensions (Figure 1A (iib)).</li>
		<li>Invert the 15 mL tube to ensure the proper mixing of rBM and cell suspension before dispensing the suspension into the plate.</li>
		<li>Transfer the rBM-containing cell suspension to a sterile reservoir and dispense 200 &#181;L/well into chilled ultra-low attachment 96-well plates using a multichannel pipette (Figure 1A (iii)). 
		NOTE: If working with several cell suspensions (e.g., more than one cell line), it is essential to dispense each cell suspension immediately after rBM addition to prevent premature gelling.</li>
		<li>Centrifuge the plate for 15 min at 750 x g&#160;using &#39;soft decent&#39;/no braking (if possible, centrifuge at 4 &#176;C to keep the rBM fluid longer but not a requirement for successful spheroid formation), to ensure that the cells are clustered together when the rBM hardens, facilitating the formation of one single spheroid.</li>
		<li>Incubate the plate in an incubator (37 &#176;C, 5% CO2, 95% humidity).</li>
		<li>Every 2-3 days acquire light microscopic images for evaluation of spheroid growth.</li>
		<li>Every 2-3 days replace 100 &#181;L of medium (remove 100 &#181;L and replace with 100 &#181;L of fresh medium).</li>
	</ol>
	</li>
	<li>Hanging drop spheroids.
	<ol>
		<li>Perform step 1.1.1-1.1.4.</li>
		<li>Dilute cells to obtain a suitable dilution. A practical dilution is 50,000 cells/mL.</li>
		<li>Remove the lid of a 10 cm2 cell culture dish and place it so it faces upwards. Add 6 mL of 1x PBS to the dish (Figure 1B (i)).</li>
		<li>Pour the cell suspension into a sterile reservoir and carefully place up to 30 drops of 40 &#181;L of cell suspension onto the lid of the cell culture dish using a multichannel pipette (Figure 1B (ii)), resulting in a concentration of 2,000 cells/drop. Avoid placing the drops too close to the edge of the lid as these drops are more likely to lose surface tension when inverting the lid in the following step.</li>
		<li>Invert the lid in a quick but controlled movement and place it on top of the 1x PBS-containing cell culture dish (Figure 1B (iii)).</li>
		<li>Place the dish in an incubator at 37 &#176;C with 5% CO2 and 95% humidity without disturbing the drops and leave them to grow for 4-6 days.</li>
		<li>If to be used for protein lysates or embedding, pool spheroids by removing the lid and tilt it, in order to wash down the drops with 1 mL of heated medium. Transfer the resulting medium&#160;containing spheroids to a 1.5 mL tube and allow them to settle to the bottom of the tube. Proceed as described in 4.4 and 6.2.2 for protein lysates and embedding, respectively.</li>
	</ol>
	</li>
</ol>
2. Drug Treatment of Spheroids
NOTE: Long-term drug treatment can be applied to the spheroids in order to screen for effects of a drug of interest. Before initiating the drug treatment, it is advisable to perform a dose response experiment of the drug(s), in order to find an appropriate dose for the experimental treatment. The doses should be based on the determined IC50/Ki of the drug and range from around 0.2x-10x of this value.
<ol>
	<li>Set up 6-12 spheroids per the desired condition as described in 1.2 or 1.3 and place in the incubator (37 &#176;C, 5% CO2, 95% humidity) for 2 days.</li>
	<li>On day 2, take light microscopic images of the spheroids.</li>
	<li>Prepare the first treatment doses (after acquiring images). 
	NOTE: The first treatment concentration must be twice the desired final concentration as the solution will be diluted 1:2 upon addition to the well containing 100 &#181;L medium. Suggested drug treatment intervals (will depend on drug half-life): Day 2, 4 and 7.</li>
	<li>Using a multichannel pipette, gently remove 100 &#181;L of medium and replace it with 100 &#181;L of drug containing medium.</li>
	<li>Place the 96-well plate back in the incubator at 37 &#176;C with 5% CO2 and 95% humidity and repeat 2.3 and 2.4 on the chosen days of treatment but now without doubling the dose to obtain correct final dose.</li>
	<li>On the final day of the protocol/treatment schedule, one or several of the following assays can be performed.</li>
</ol>
3. Cell Viability Assay for Spheroids
<ol>
	<li>Set up 4-6 spheroids per the desired condition as described in 1.2 or 1.3 and place in the incubator at 37 &#176;C with 5% CO2 and 95% humidity. 
	NOTE: In this case, the cell viability assay was performed on day 7 or 9, after having monitored spheroid growth every 2-3 days by light microscopy as described above (point 1.2.5 and 1.3.13).</li>
	<li>Thaw the viability assay reagent (see Table of Materials) and let it equilibrate to RT prior to use.</li>
	<li>Mix gently by inverting to obtain a homogeneous solution.</li>
	<li>Before performing the assay, remove 50% of the culture medium from the spheroids (100 &#181;L).</li>
	<li>Add cell viability reagent to each well at a 1:3 ratio to the amount of medium present in the well (Figure 2A (i)) For a 96-well plate, add 50 &#181;L of reagent to 100 &#181;L of medium.</li>
	<li>Mix the contents vigorously for 5 min to induce cell lysis (Figure 2A (ii)).</li>
	<li>Incubate for 25 min at RT to stabilize the luminescent signal (Figure 2A (iii)).</li>
	<li>Record the luminescent signal (Figure 2A (iv)).</li>
</ol>
4. Preparing Protein Lysates for Western Blotting from 3D Spheroid Cultures
NOTE:&#160;When collecting the spheroids, it is advisable to use a P200 pipette and cut the end of the tip to allow a bigger opening and hence an easier capture of the spheroids without disturbing their structure.
<ol>
	<li>For each condition, pool a minimum of 12, ideally 18-24 spheroids (depending on spheroid size) in a 1.5 mL tube (avoid 2 mL tubes, as the next steps will become more difficult due to their less pointy bottom). 
	NOTE:&#160;If the amount of medium exceeds 1.5 mL before having collected all the spheroids, allow the collected spheroids to settle at the bottom (happens very quickly, centrifugation not necessary) and discard half the volume of the tube before continuing collecting the remaining spheroids.</li>
	<li>Place tubes on ice and allow the spheroids to settle at the bottom of the 1.5 mL tube.</li>
	<li>Move from the sterile cell laboratory to the regular laboratory.</li>
	<li>Wash spheroids twice in 1 mL of ice-cold 1x PBS. Let spheroids settle before removing 1x PBS between each washing step.</li>
	<li>Aspirate as much 1x PBS as possible without disturbing or removing the spheroids.</li>
	<li>Add 5 &#181;L of heated lysis buffer (LB) with phosphatase- and protease inhibitors, per spheroid (e.g., 10 spheroids = 50 &#181;L LB).</li>
	<li>Repeat intervals of vortex followed by spin down until spheroids are dissolved. Perform a cycle of vortexing for 30 s followed by centrifugation (a quick spin using a tabletop centrifuge is sufficient) for 10 s for approx. 5-10 min depending on the size and the compactness of the spheroids. 
	NOTE:&#160;The protocol can be paused here. Keep the lysates at -20 &#176;C until proceeding with sonication, homogenization and protein determination as in a standard 2D protein lysate protocol, followed by western blotting using standard protocols.</li>
</ol>
5. Propidium Iodide (PI) Staining of Spheroids
<ol>
	<li>Set up 3-6 spheroids per desired condition as described in 1.2 or 1.3 and place in the incubator at 37 &#176;C with 5% CO2 and 95% humidity.</li>
	<li>In a sterile cell culture lab, heat 1x PBS to 37 &#176;C.</li>
	<li>Make a PI solution of 4 &#181;M by diluting stock solution in 1x PBS: Dilute a 1 mg/mL aqueous stock of PI 1:350 in 1x PBS. 
	NOTE: This concentration will be further halved upon addition of the solution to the wells giving a final concentration of 2 &#181;M. 100 &#181;L of this solution is needed for each well containing a spheroid. 
	CAUTION: Propidium iodide (PI) must be handled in a fume hood and wearing gloves. PI is light sensitive. Protect from light when handling.</li>
	<li>Remove 100 &#181;L of the medium from each well in the 96-well plate without removing the spheroids.</li>
	<li>Wash out the remaining medium by adding 100 &#181;L of heated 1x PBS to all wells followed by removing 100 &#181;L of the liquid in the wells. Repeat this washing step 3 times.</li>
	<li>Add 100 &#181;L of the PI solution to each well, cover the plate in aluminum foil and place it in an incubator at 37 &#176;C with 5% CO2 and 95% humidity for 10-15 min.</li>
	<li>Repeat the 3 washing steps described in 4.5 to wash out PI solution, in order to diminish background signal when imaging.</li>
	<li>Use an epifluorescence microscope to image the spheroids. To evaluate the viability of cells in the spheroid core take z-stacks to get images with varying depths of the spheroid. 
	NOTE: A step size around 18-35 &#181;m between each slice depending on spheroid size is advisable, giving approximately 11-18 stacks per spheroid. Z-stacks can be processed in ImageJ using the z-projection function, which can combine all z-stacks into one final picture, giving an overview of the staining throughout the spheroid (for further guidelines on the use of ImageJ for this purpose, see (https://imagej.net/Z-functions).</li>
</ol>
6. Embedding of 3D Spheroids
<ol>
	<li>Prepare the agarose gel into which the spheroids are embedded (only necessary first time performing the protocol).
	<ol>
		<li>Mix 1 g of bactoagar in 50 mL of ddH2O.</li>
		<li>Heat slowly in microwave oven until the bactoagar has dissolved and a homogenous gel has formed. Do not allow the gel to boil.</li>
		<li>Keep the bactoagar warm in a water bath at 60 &#176;C.</li>
		<li>Keep at 4 &#176;C between experiments.</li>
	</ol>
	</li>
	<li>Embedding of spheroids.
	<ol>
		<li>On day 1, for each condition, pool a minimum of 12 spheroids in a 1.5 mL tube.</li>
		<li>Wash once with 1 mL of ice-cold 1x PBS.</li>
		<li>To fix the spheroids, add 1 mL of 4% paraformaldehyde. 
		NOTE: Handling of paraformaldehyde should be performed in a fume hood.</li>
		<li>Let them incubate for 24 h at RT.</li>
		<li>On day 2, heat the agarose gel carefully by placing it in a water-filled beaker in a microwave oven. Ensure that the gel does not boil! Keep warm in a benchtop heating plate, at 60 &#176;C until use.</li>
		<li>Wash spheroids twice with 1 mL of ice-cold 1x PBS.</li>
		<li>Aspirate most of the 1x PBS (leaving approximately 100 &#181;L at this point is practical for handling the spheroids).</li>
		<li>Prepare a 20 &#181;L pipette by cutting the pipette tip at an incline to obtain a pointier tip with a larger hole (see illustration). 
		NOTE: The next part has to be done quickly to ensure optimal spheroid transfer and to avoid solidification of gel drop. If no heating block is available, it is recommended to first catch the spheroids and then make the agarose drop (i.e., switching the order of points 6.2.9 and 6.2.10).</li>
		<li>Make an agarose gel drop on a microscope slide. Place the slide on a warm heating block to prevent the agarose from solidifying.</li>
		<li>Using the modified pipette tip (see 6.2.8), catch as many spheroids as possible in a volume of 15-20 &#181;L.</li>
		<li>Carefully inject the 15-20 &#181;L spheroid-containing 1x PBS into the center of the agarose gel drop without touching the microscope slide. 
		NOTE: This is a slightly difficult point. The spheroids will be lost if the pipette tip touches the microscope slide when injecting the spheroids into the gel drop. It is advisable to practice the whole process of making the agarose drop and injecting the spheroids by injecting a colored liquid into the drop. This will allow visualization of a potential penetration through the drop, as the colored liquid will be leaking out onto the slide.</li>
		<li>Let the agarose gel drop harden by incubating for 5-10 min at RT or at 4 &#176;C. Once the gel drop has solidified somewhat (but is still rather soft), carefully push the gel drop from the microscope slide into a plastic tissue cassette with a scalpel.</li>
		<li>Cover the plastic tissue cassettes in 70% ethanol. 
		NOTE: At this point the spheroids can be used directly or stored for months.</li>
		<li>Embed the agarose-embedded spheroid in paraffin, section into 2-3 &#181;m thick layer slides and stain with hematoxylin and eosin or subject to immuno-histological staining.</li>
	</ol>
	</li>
</ol>

The authors declare no conflict of interest.

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

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27.3K Views.  University of Copenhagen. 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.

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

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 ...

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.

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

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Biochemistry

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...