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 and chemical cell-cell interactions9,17. For example, the acidity of extracellular pH, which can reach values as low as 6-6.5 in many solid tumors25,26,27,28,29, causes weakly basic chemotherapeutic compounds, such as doxorubicin, mitoxantrone and the zwitterion paclitaxel, to be charged. This reduces their uptake into the tumor cells and can influence the activity of multidrug resistance proteins such as p-glycoprotein30,31,32. Also the cell proliferation, which is pivotal to the effect of most chemotherapeutic compounds, is generally reduced in 3D compared to 2D conditions and hence is likely better mimicked in tumor spheroids than in 2D cell culture8,33,34. Finally, the dense tumor microenvironment is the origin of numerous physical and soluble signaling cues directing intracellular signaling pathways regulating cell growth, survival and death. Thus, when analyzing drug efficacy, 3D culture systems are a pivotal step before embarking on in vivo models. A major drawback of 3D culture is, however, the increased complexity of analysis compared to that of 2D culture. We have described here simple and relatively inexpensive techniques for spheroid formation using a variety of cancer cell types. We have shown examples of how spheroid formation must be optimized for each cell type studied and have described how to obtain quantitative data on cell viability, cell death, and associated signaling pathways, in such spheroids. There is no obvious growth- or morphological differences between the three models described here. In our hands, the variation in morphology may be slightly greater using the hanging drop method, yet an advantage of this method is that rBM is not needed. We have focused here on spheroids produced from a single cancer cell type. The spheroid model is, however, also amenable to co-culture, for instance of cancer cells with fibroblasts, monocytes/macrophages, endothelial cells, and/or adipocytes35,36,37. Other advanced applications of this model include the combination with 3D printed fluidic devices allowing dosing through a semipermeable membrane, followed by harvesting for quantitative proteomic profiling38.
While, as noted above, the phenotype of cells grown in 3D spheroids generally mimics that of in vivo tumors much better than do cells grown in 2D, the extent to which such spheroids are in fact relevant models of the corresponding in vivo tumors is dependent on numerous factors and has to be carefully evaluated. Parameters which will impact how well such spheroids mimic the in vivo condition include the cellular composition of the tumor and its relative ECM composition. For instance, the rBM which we have employed as ECM in the protocols provided here is a good choice for mimicking early stages of epithelial cancers, around the time of breaching the basement membrane, other ECM compositions will be more relevant for certain tumor types and -stages. Furthermore, the capacity for cell-cell adhesion differs widely between cancer cell lines, depending on their expression of cell-cell and cell-matrix adhesion proteins such as cadherins and integrins22.&#160;
As described here, spheroid growth and morphology can easily and non-invasively be monitored every 2-3 days using a light microscope with low magnification optics and a large field of view. However, because the cytotoxic stress, such as chemotherapy treatment, affects spheroid morphology very differently and, in a manner, depending on the cell type and treatment scheme, it is not enough to rely on the morphology and circumference alone for evaluating treatment effect. For instance, spheroids may become looser with treatment and emerging cell death, or all death may occur in the necrotic core, while the surface is not detectably affected. In both cases, the result may be an erroneous impression that the number of live cells in the spheroid is not reduced by the treatment. Quantitative- and whole-spheroid techniques are therefore essential for evaluating treatment effect. For quantitative evaluation of cell death, the acid phosphatase assay, which as the name implies measures the activity of cytosolic acid phosphatase has been employed21. However, in our hands, while this assay generally nicely reflects the number of cells seeded, it does not adequately capture rapid treatment-induced cell death (data not shown), likely because the acid phosphatase remains active for some time after cell death. Furthermore, this assay requires complete removal of the medium, which increases error especially with fragile, chemotherapy-treated spheroids. The cell viability assay described here, which is based on cellular ATP content, was chosen based on its simple and time efficient protocol and high reproducibility. Furthermore, this assay does not require complete removal of culture medium which is an advantage when working with spheroids. As shown in representative results, this assay captures well both cell number and expected chemotherapy treatment effects. However, a pitfall of this technique is, obviously, that metabolic changes reducing intracellular ATP content may erroneously be recorded as a lower cell number. Hence, parallel assessment of spheroid volume and morphology, or PI staining, is advisable to validate results.
Spheroid lysis followed by western blotting can provide semi-quantitative insight into the state of signaling processes, cell death-, growth- and viability pathways. The use of western blotting is complicated when rBM is used to prepare the spheroids, since this will comprise a substantial fraction of the lysate protein content, and more importantly, its fractional contribution will increase with decreasing cellular content during chemotherapeutic cell death. It is in principle possible to remove the rBM by centrifugation; however, this is a critical step, as it is difficult to completely remove all rBM, and this will preclude quantitative comparison between conditions. For such spheroids, and in general for spatially resolved assessment of death pathways and relevant signaling parameters, embedding and IHC are strong tools. Other approaches may be considered: live confocal imaging of (relatively small) intact spheroids39. Another interesting property of spheroids is that given their rather regular &#34;ball&#34;&#160;shape, they lend themselves well to iteration between mathematical modeling and wet lab experiments, to increase the understanding of the importance of the above-mentioned gradients of oxygen, pH, and nutrients within spheroids, and, by extrapolation, tumors40,41. Thus, although important 3D tumor models of much greater complexity are emerging, including a wide range of organotypic and organoid cultures based on complex biological as well as inert scaffolds, and, not least, patient-derived xenografts42, spheroids remain an important tool because of their superior biological relevance compared to 2D culture, combined with relative ease of handling.&#160;
In summary, we present here a series of simple methods for analysis of anti-cancer treatment-induced changes in cancer cell viability and death in 3D culture. The composition of the spheroids can be modified depending on the properties and biology of the cells employed, and the quantitative and qualitative analyses presented are useful both for assessing dose-response relationships and for gaining insight into the signaling- and death pathways involved.

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

<table border="0" cellpadding="0" cellspacing="0" style="width:1292px;" width="1292">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI)</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:264px;"># C10595&#160;</td>
			<td style="width:336px;">For staining nuclei</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">5-Fluorouracil (5-FU)</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:264px;">#F6627</td>
			<td style="width:336px;">Component in chemotherapeutic treatment</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">5-(N-ethyl-isopropyl) amiloride (EIPA)</td>
			<td style="width:284px;">Life Technologies</td>
			<td style="width:264px;">#E3111</td>
			<td style="width:336px;">Inhibitor of NHE1</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Antibody against PARP and cPARP</td>
			<td style="width:284px;">Cell signaling</td>
			<td>#9542</td>
			<td style="width:336px;">Used in western blotting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Antibody against Ki-67</td>
			<td style="width:284px;">Cell signaling</td>
			<td style="width:264px;">#9449</td>
			<td style="width:336px;">Used for IHC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Antibody against p53</td>
			<td style="width:284px;">Cell Signaling&#160;</td>
			<td style="width:264px;">#2524&#160;</td>
			<td style="width:336px;">Used for IHC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Antibody against &#946;-actin</td>
			<td style="width:284px;">Sigma&#160;</td>
			<td style="width:264px;">A5441</td>
			<td style="width:336px;">Used in western blotting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Bactoagar</td>
			<td style="width:284px;">BD Bioscience</td>
			<td style="width:264px;">#214010</td>
			<td style="width:336px;">Used for agarose gel preparation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Benchmark protein ladder</td>
			<td style="width:284px;">Invitrogen</td>
			<td>#10747-012</td>
			<td style="width:336px;">Used for SDS-PAGE</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Bio-Rad DC Protein Assay kit</td>
			<td>Bio-Rad Laboratories</td>
			<td>#500-0113, #500-0114, #500-0115&#160;&#160;</td>
			<td style="width:336px;">Used for protein determination from lysates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">B&#252;rker chamber</td>
			<td style="width:284px;">Marienfeld</td>
			<td style="width:264px;">610311</td>
			<td style="width:336px;">For cell counting&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">BX63 epifluoresence microscope</td>
			<td style="width:284px;">Olympus</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Used for fluorescent imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">CellTiter-Glo 3D Cell Viability Assay</td>
			<td style="width:284px;">Promega</td>
			<td style="width:264px;">#G9681</td>
			<td style="width:336px;">Used for the cell viability assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:408px;">Cisplatin</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:264px;">#P4394&#160;</td>
			<td style="width:336px;">Component in chemotherapeutic treatment</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:408px;">Corning Spheroid Microplate, 96 well, Black with clear round bottom,&#160; Ultra-low attachment, With lid, Sterile</td>
			<td style="width:284px;">Corning</td>
			<td style="width:264px;">#4520</td>
			<td style="width:336px;">Used for growing spheroids with luminescence measurements as end point</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:408px;">Corning 96 well, clear round bottom,&#160; Ultra-low attachment microplate, With lid, Sterile</td>
			<td style="width:284px;">Corning</td>
			<td style="width:264px;">#7007</td>
			<td style="width:336px;">Sufficient for spheroid growth without luminescence measurements as end point</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Criterion TGX Precast Gels</td>
			<td style="width:284px;">Bio-Rad</td>
			<td style="width:264px;">5671025</td>
			<td style="width:336px;">Used for SDS-PAGE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Doxorubicin</td>
			<td style="width:284px;">Abcam</td>
			<td style="width:264px;">#120629</td>
			<td style="width:336px;">Component in chemotherapeutic treatment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">FLUOStar Optima Microplate reader</td>
			<td style="width:284px;">BMG Labtech</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Used for recording luminescence&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:408px;">Formaldehyde&#160;</td>
			<td style="width:284px;">VWR Chemicals&#160;</td>
			<td style="width:264px;">#9713.1000&#160;</td>
			<td style="width:336px;">Used for cell fixation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:408px;">Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td style="width:284px;">Gibco</td>
			<td style="width:264px;">#A1413202</td>
			<td style="width:336px;">Keep at 4 &#176;C to prevent solidification. Referred to as rBM in the protocol.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:408px;">Heat-inactivated FBS</td>
			<td>Sigma</td>
			<td style="width:264px;">#F9665</td>
			<td style="width:336px;">Serum for growth media</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:408px;">ImageJ</td>
			<td style="width:284px;">NIH</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Scientific Image analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Medim Uni-safe casette</td>
			<td>Medim Histotechnologie</td>
			<td>10-0114</td>
			<td style="width:336px;">Used for storage of embedded spheroids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Mini protease inhibitor cocktail tablets</td>
			<td style="width:284px;">Roche Diagnostics GmBH&#160;</td>
			<td style="width:264px;"># 11836153001</td>
			<td style="width:336px;">Used for lysis buffer preparation</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">MZ16 microscope</td>
			<td style="width:284px;">Leica</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Used for light microscopic images</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">NuPAGE LDS 4x Sample Buffer&#160;</td>
			<td>Invitrogen</td>
			<td>#NP0007</td>
			<td style="width:336px;">Used for western blotting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pierce ECL Western blotting substrate</td>
			<td style="width:284px;">Thermo scientific</td>
			<td>#32106</td>
			<td>Used for western blotting</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ponceau S</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td>#P7170-1L</td>
			<td style="width:336px;">Used for protein band staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Prism 6.0</td>
			<td style="width:284px;">Graphpad</td>
			<td style="width:264px;"></td>
			<td style="width:336px;">Scientific graphing and statistical software</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:408px;">Propidium iodide (1mg/ml solution in water)</td>
			<td>Invitrogen&#160;</td>
			<td>P3566</td>
			<td>Light sensitive&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:408px;">Sterile reservoirs, multichannel</td>
			<td style="width:284px;">SPL lifesciences</td>
			<td style="width:264px;">21002</td>
			<td style="width:336px;">Used for seeding cells for spheroid formation</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:408px;">Superfrost Ultra-Plus Adhesion slide&#160;</td>
			<td style="width:284px;">Menzel-Gl&#228;ser</td>
			<td style="width:264px;">#J3800AMNZ</td>
			<td style="width:336px;">Microscope glass slide used for embedding</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:408px;">Tamoxifen</td>
			<td style="width:284px;">Sigma-Aldrich</td>
			<td style="width:264px;">#T5648</td>
			<td style="width:336px;">Used as chemotherapeutic treatment</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Trans-blot Turbo 0.2 &#181;m nitrocellulose membranes</td>
			<td style="width:284px;">Bio-Rad</td>
			<td>#170-4159</td>
			<td style="width:336px;">Used for western blotting</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:408px;">Tris/Glycine/SDS running buffer&#160;</td>
			<td style="width:284px;">Bio-Rad&#160;</td>
			<td>#161 0732</td>
			<td style="width:336px;">Used for SDS-PAGE</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:408px;">Trypsin-EDTA solution</td>
			<td style="width:284px;">Sigma</td>
			<td>#T4174&#160;</td>
			<td style="width:336px;">Cell dissociation enzyme</td>
		</tr>
	</tbody>
</table>

assessing cell viability and death in 3d spheroid cultures of cancer cells

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

Spheroid growth assays based on the spheroid formation protocol schematically illustrated in Figure 1A and Figure 1B, were used as a starting point for analysis of the effects of anti-cancer drug treatments in a 3D tumor mimicking setting. The ease with which spheroids are formed is cell line specific, and some cell lines require supplementation with rBM in order to form coherent spheroids22. The concentration of rBM added can profoundly affect the morphology of the spheroids. As shown in Figure 1C and Figure 1D, varying the concentration of rBM between 0 and 4% alters the compactness and morphology of the spheroids in a cell type dependent manner. Figure 1C demonstrates how the addition of up to 2.5% rBM allows spheroid formation in SKBr-3 breast cancer cells, with no further effect at concentrations above 2.5% rBM. In contrast, BxPC3 pancreatic ductal adenocarcinoma (PDAC) cells, which exhibit an epithelial morphology, spontaneously form small, compact spheroids (Figure 1D, upper, left panel). In this cell type, increasing rBM concentration to 1.5% or above elicits a distinct morphological change from spheroid to more convoluted structures with protrusions and invaginations, reminiscent of ductal tubular structure formation. Conversely, the addition of rBM to two other PDAC cell lines, MiaPaCa and Panc-1, which have a more mesenchymal phenotype, allows the loose cellular aggregates to become tighter and form more compact spheroids (Figure 1D, middle and lower panels). These results show that the precise amount of rBM resulting in optimal spheroid formation must be titrated for each cell line and condition.
A quantitative assessment of cell viability within the spheroids upon drug treatment was necessary to evaluate the effect of anti-cancer drug treatments. The assay described here is a luciferin-luciferase-based assay, which measures ATP released from live cells within spheroids. The principle of the assay is illustrated in Figure 2A. The luminescent signal generated in this assay is easily recorded by a plate reader (Figure 2A) and correlates well with viability measured by other methods23. The linear relation between ATP concentration and luminescence in the relevant concentration range is shown in Figure 2B, while Figure 2C shows the ability of the assay to assess cell death in 3D spheroids treated with anti-cancer therapy. In order to further evaluate the linearity of the assay in the relevant range, experiments to establish standard curves of the luminescent signal as a function of the number of cells were carried out (Figure 2D and Figure 2E). These results indicate that the assay is suitable for estimating cell viability in 3D spheroid cultures and that it is applicable for investigating drug-induced loss of cell viability.
A combination of light microscopic images acquired every two to three days, during the treatment period and a final quantitative assessment of cell viability allows close supervision of spheroid growth and morphology as well as the assessment of optimal treatment dose. The latter is exemplified in Figure 3A and Figure 3B, where a dose-response experiment was performed to determine the dose necessary for 50% reduced cell viability in MDA-MB-231 breast cancer spheroids. Treatment effects on spheroid morphology are visualized in Figure 3C and Figure 3D for MDA-MB-231 and MCF-7 spheroids, respectively. During treatment with the chosen chemotherapeutic cocktail, the compactness of MDA-MB-231 spheroids increases, while during treatment with tamoxifen, MCF-7 spheroids become increasingly frayed and uneven. In both cases, a clear drop in cell viability is visible after 7 (MDA-MB-231) or 9 (MCF-7) days of treatment (Figure 3E and Figure 3F). This demonstrates the need for both a visual and a quantitative assessment of treatment-mediated effects on spheroid cell viability and morphology as well as that these parameters are highly cell- and treatment-type specific.&#160;&#160;
As a supplement to the cell viability assay, staining of dead cells with PI, which cannot cross the membrane and therefore only stains necrotic or late apoptotic cells with compromised membrane integrity, allows for a quick spatial evaluation of dead cells in response to treatment, without the time-consuming protocol of embedding, sectioning and IHC. As illustrated in Figure 4A the spatial arrangement of dead cells upon an increasing concentration of an inhibitor, in this case, the Na+/H+ exchanger 1 (NHE1) inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), can be visualized. As seen, control spheroids show a limited necrotic/late apoptotic core, whereas the dead cells are distributed throughout the spheroid as the concentration of EIPA is increased.
In order to quantify the relative induction of apoptotic stress following different treatments, spheroids were lysed and subjected to SDS-PAGE gel electrophoresis and western blotting for full-length and cleaved poly (ADP-ribose) polymerase (PARP). Representative results are shown in Figure 4B and Figure 4C. In this experiment, spheroids were prepared from MDA-MB-231 cells in which the lactate-proton cotransporter MCT4 or the Na+, HCO3- cotransporter NBCn1 were knocked down using siRNA. The knockdown was evaluated by western blotting for MCT4 and NBCn1 (unpublished data). As seen, the knockdown of MCT4, but not of NBCn1, robustly increases PARP cleavage, consistent with our previous demonstration that stable knockdown of MCT4 in MDA-MB-231 cells decreases tumor growth in vivo24.
To further analyze the effects of treatment and obtain information on the specific signaling-, growth arrest, and death pathways activated, the spheroids can in addition to western blot analysis be embedded and subjected to immunohistochemistry (IHC) analysis. IHC analysis of the spheroid sections allows the use of specific antibodies or markers of cell proliferation, cell cycle and programmed cell death, and facilitates a visualization of the spatial arrangement of proliferative and apoptotic cells in the spheroid.
A schematic figure of the embedding protocol for IHC analysis of spheroids is presented in Figure 5A. A representative light microscopic image of an approx. 3 &#181;m thick microtome section of an embedded spheroid is shown in Figure 5B, and an immunofluorescence image of a spheroid stained for the tumor suppressor protein p53 (nuclei stained using DAPI), is shown as Figure 5C. Examples of DMSO and chemotherapy-treated spheroids stained for the cell proliferation marker Ki-67 or for p53 are shown in Figure 5D and Figure 5E, respectively. Consistent with the antiproliferative effect of the chemotherapy treatment, the number of Ki-67 positive cells are greater in the DMSO control than in the chemotherapy-treated spheroid (Figure 5D). In contrast, p53 expression is increased during conditions of cell stress, apoptosis and growth arrest, and consequently, the number of p53-stained cells is substantially higher in the chemotherapy-treated spheroids compared to DMSO controls (Figure 5E).
These results illustrate examples of how spatially resolved (PI staining, IHC) or quantitative (western blotting) information on drug treatment effects in 3D spheroids can be obtained.&#160;&#160;&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59714/59714fig1.jpg" /> 
Figure 1: Spontaneous and rBM-mediated spheroid formation. (A) Schematic representation of spheroid formation using ultra-low attachment 96-well round bottom plates, with optional use of rBM. Individual steps marked by (i-iii). (B) Schematic representation of spheroid formation using the hanging drop method. Individual steps are marked by (i-iii) (C) Representative images of rBM-mediated spheroid formation of SKBr-3 cells. Cells were seeded in ultra-low attachment 96-well round bottom plates with increasing concentrations of rBM and grown for 9 days. Scale bar&#160;100 &#181;m. (n=3). (D) Representative images of BxPC-3, MiaPaCa and Panc-1 cells seeded for spheroid formation in ultra-low attachment 96-well round bottom plates with concentrations of rBM from 0.5-2.5 %. Spheroids were grown for 4 days. Scale bar = 250 &#181;m. (n=3). <a href="https://www.jove.com/files/ftp_upload/59714/59714fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59714/59714fig2.jpg" /> 
Figure 2: Principle and evaluation of the cell viability assay. (A) Schematic representation of the 3D cell viability assay. Individual steps denoted by (i-iv). (B) Luminescent signal as a function of ATP concentration. Dilutions of ATP were plated in a 96-well plate and cell viability reagent added to each well. Luminescence was recorded after 30 min at 405 nm. 1 n. (C) Viability, measured as luminescence, of control and chemotherapy-treated MCF-7 spheroids. MCF-7 cells were seeded in ultra-low attachment round-bottom plates and were grown for 7 days. Chemotherapy treatment (5 &#956;M Cisplatin, 5 &#956;M Doxorubicin and 30 nM 5-FU) was applied on day 2 and 4. Bars represent mean values with SD. 1 n. (D) Luminescent signal as function of the number of MCF-7 cells seeded. MCF-7 cells were seeded in 96-well plates at the indicated cell number and allowed to grow for 48 h, after which cell viability was measured. Error bars represent SD. 1 n. (E) As described in D for MDA-MB-231 cells. <a href="https://www.jove.com/files/ftp_upload/59714/59714fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59714/59714fig3.jpg" /> 
Figure 3: Effects of treatment regimens on spheroid morphology and cell viability. (A) Representative images of MDA-MB-231 spheroids on day 2, 4 and 7. MDA-MB-231 cells were seeded in ultra-low attachment round bottom 96-well plates. Treatment with increasing doses of chemotherapy was started on day 2, at which time all spheroids were of similar size. Rows show spheroids at increasing doses of chemotherapy, and columns show spheroids representative of size at day 2, 4, and 7 at the indicated dose. The lowest dose was 18.75 nM Cisplatin, 18.75 nM Doxorubicin, 0.0625 nM 5-Fluorouracil (5-FU) and this dose was doubled for each image shown, resulting in a maximal dose of 0.3 &#181;M Cisplatin, 0.3 &#181;M Doxorubicin and 2 nM 5-FU. Scale bar = 100 &#181;m. (2 n). (B) Viability of MDA-MB-231 spheroids, measured as luminescence, after 7 days of chemotherapeutic treatment. The bars represent mean values with SEM. 2 n. (C,D) Representative images of MDA-MB-231 (C) and MCF-7 spheroids (D) on day 2, 4, 7 and for MCF-7 spheroids 9. Cells seeded as in (A) and treated with either chemotherapy (Chemo, 18.75 nM Cisplatin, 18.75 nM Doxorubicin, 0.0625 nM 5-FU) on day 2 and 4 (C) or with 2 &#181;M Tamoxifen (Tam) on day 2, 4 and 7 (D). Scale bar = 100 &#181;m. (4 n and 3 n), respectively. (E,F) Viability, measured as luminescence, on day 7 and 9 for (C) and (D), respectively. To test for statistically significant difference between conditions an unpaired Student&#8217;s t-test was performed. **** denotes p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/59714/59714fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59714/59714fig4.jpg" /> 
Figure 4: Propidium iodide staining and western blot analysis of spheroids. (A) Representative images of PI-stained MCF-7 spheroids after 9 days of treatment. MCF-7 cells were seeded in ultra-low attachment 96-well plates grown for 9 days and treated with increasing concentrations of EIPA on day 2, 4 and 7. On day 9, the spheroids were stained with PI and images were acquired on an epifluorescence microscope. Scale bar = 200 &#181;m. (1 n.) (B) Representative western blots of MDA-MB-231 cells after knockout/knockdown of acid-base transporters. NHE1 was knocked out by CRISPR/Cas9 in MDA-MB-231 cells 12 and the cells were subsequently transiently transfected with siRNA against MCT4 or NBCn1, and grown as spheroids for 9 days before being lysed and subjected to western blotting using an antibody recognizing total and cleaved (c)PARP. (C) Quantification of the ratio of cPARP to PARP protein level, normalized to loading control (&#946;-actin). (1 n). <a href="https://www.jove.com/files/ftp_upload/59714/59714fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59714/59714fig5.jpg" /> 
Figure 5: Fixing, embedding and immunohistochemistry analysis of spheroids. (A) Schematic representation of the protocol for embedding of spheroids. Individual steps are marked as (i-vii). (B) Image of embedded MDA-MB-231 spheroid. Scale bar: 50 &#181;m. (C) Representative image of chemotherapy-treated MDA-MB-231 spheroid subjected to IHC analysis with antibodies against p-53. Dashed lines show the circumference of the spheroid. Scale bar = 20 &#181;m. (D,E) Representative images of DMSO- or chemotherapy-treated (upper and lower panels, respectively) MDA-MB-231 spheroids. MDA-MB-231 cells were seeded in ultra-low attachment 96-well plates, grown for 7 days and treated with chemotherapy on day 2 and 4. On day 7, the spheroids were embedded followed by analysis by IHC with primary antibodies against Ki-67 (D) and p53 (E). White boxes represent zoom images. Scale bar = 20 &#181;m in both magnifications, (n=3). <a href="https://www.jove.com/files/ftp_upload/59714/59714fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

27.0K 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

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 ...

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 ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

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 ...