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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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. 

Protocol

1. Generation of Spheroids

  1. 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 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.
    1. Grow cells as monolayer until 70-80% confluency.
    2. 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 °C in 5% CO2 and 95% humidity.
    3. 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.
    4. Use a Bürker 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.
  2. Spontaneous spheroid formation
    1. Perform steps 1.1.1-1.1.4.
    2. 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)).
    3. Fill the outer ring of wells with 1x PBS or growth medium to reduce evaporation from the remaining wells. Transfer the cell suspension to a sterile reservoir and, using a multichannel pipette, dispense 200 µL/well into ultra-low attachment 96-well round bottom plates (Figure 1A (iii)).
    4. Incubate the plate in an incubator at 37 °C with 5% CO2, 95% humidity.
    5. 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.
    6. Every 2-3 days (after acquiring images) replace 100 µL of medium (remove 100 µL of the spent medium and replace with 100 µL 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.
  3. 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 °C. Thaw the rBM on ice either overnight at 4 °C or 2-4 h at room temperature (RT) before plating.
    1. Thaw rBM on ice (see Table of Materials).
    2. Keep plates and reservoirs (if individually wrapped) on ice before use.
    3. Perform steps 1.1.1-1.1.4.
    4. 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)).
    5. Place the 15 mL tube containing the diluted cell suspension on ice (e.g., in glass beaker) (Figure 1A (iia)).
    6. 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.
    7. Resuspend rBM gently to ensure a homogenous gel.
    8. Add 1-2% rBM (optimal concentration needs to be determined for each cell line) to the chilled cell suspensions (Figure 1A (iib)).
    9. Invert the 15 mL tube to ensure the proper mixing of rBM and cell suspension before dispensing the suspension into the plate.
    10. Transfer the rBM-containing cell suspension to a sterile reservoir and dispense 200 µL/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.
    11. Centrifuge the plate for 15 min at 750 x g using 'soft decent'/no braking (if possible, centrifuge at 4 °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.
    12. Incubate the plate in an incubator (37 °C, 5% CO2, 95% humidity).
    13. Every 2-3 days acquire light microscopic images for evaluation of spheroid growth.
    14. Every 2-3 days replace 100 µL of medium (remove 100 µL and replace with 100 µL of fresh medium).
  4. Hanging drop spheroids.
    1. Perform step 1.1.1-1.1.4.
    2. Dilute cells to obtain a suitable dilution. A practical dilution is 50,000 cells/mL.
    3. 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)).
    4. Pour the cell suspension into a sterile reservoir and carefully place up to 30 drops of 40 µL 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.
    5. 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)).
    6. Place the dish in an incubator at 37 °C with 5% CO2 and 95% humidity without disturbing the drops and leave them to grow for 4-6 days.
    7. 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 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.

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.

  1. Set up 6-12 spheroids per the desired condition as described in 1.2 or 1.3 and place in the incubator (37 °C, 5% CO2, 95% humidity) for 2 days.
  2. On day 2, take light microscopic images of the spheroids.
  3. 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 µL medium. Suggested drug treatment intervals (will depend on drug half-life): Day 2, 4 and 7.
  4. Using a multichannel pipette, gently remove 100 µL of medium and replace it with 100 µL of drug containing medium.
  5. Place the 96-well plate back in the incubator at 37 °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.
  6. On the final day of the protocol/treatment schedule, one or several of the following assays can be performed.

3. Cell Viability Assay for Spheroids

  1. Set up 4-6 spheroids per the desired condition as described in 1.2 or 1.3 and place in the incubator at 37 °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).
  2. Thaw the viability assay reagent (see Table of Materials) and let it equilibrate to RT prior to use.
  3. Mix gently by inverting to obtain a homogeneous solution.
  4. Before performing the assay, remove 50% of the culture medium from the spheroids (100 µL).
  5. 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 µL of reagent to 100 µL of medium.
  6. Mix the contents vigorously for 5 min to induce cell lysis (Figure 2A (ii)).
  7. Incubate for 25 min at RT to stabilize the luminescent signal (Figure 2A (iii)).
  8. Record the luminescent signal (Figure 2A (iv)).

4. Preparing Protein Lysates for Western Blotting from 3D Spheroid Cultures

NOTE: 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.

  1. 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: 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.
  2. Place tubes on ice and allow the spheroids to settle at the bottom of the 1.5 mL tube.
  3. Move from the sterile cell laboratory to the regular laboratory.
  4. Wash spheroids twice in 1 mL of ice-cold 1x PBS. Let spheroids settle before removing 1x PBS between each washing step.
  5. Aspirate as much 1x PBS as possible without disturbing or removing the spheroids.
  6. Add 5 µL of heated lysis buffer (LB) with phosphatase- and protease inhibitors, per spheroid (e.g., 10 spheroids = 50 µL LB).
  7. 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: The protocol can be paused here. Keep the lysates at -20 °C until proceeding with sonication, homogenization and protein determination as in a standard 2D protein lysate protocol, followed by western blotting using standard protocols.

5. Propidium Iodide (PI) Staining of Spheroids

  1. Set up 3-6 spheroids per desired condition as described in 1.2 or 1.3 and place in the incubator at 37 °C with 5% CO2 and 95% humidity.
  2. In a sterile cell culture lab, heat 1x PBS to 37 °C.
  3. Make a PI solution of 4 µM 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 µM. 100 µL 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.
  4. Remove 100 µL of the medium from each well in the 96-well plate without removing the spheroids.
  5. Wash out the remaining medium by adding 100 µL of heated 1x PBS to all wells followed by removing 100 µL of the liquid in the wells. Repeat this washing step 3 times.
  6. Add 100 µL of the PI solution to each well, cover the plate in aluminum foil and place it in an incubator at 37 °C with 5% CO2 and 95% humidity for 10-15 min.
  7. Repeat the 3 washing steps described in 4.5 to wash out PI solution, in order to diminish background signal when imaging.
  8. 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 µm 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).

6. Embedding of 3D Spheroids

  1. Prepare the agarose gel into which the spheroids are embedded (only necessary first time performing the protocol).
    1. Mix 1 g of bactoagar in 50 mL of ddH2O.
    2. Heat slowly in microwave oven until the bactoagar has dissolved and a homogenous gel has formed. Do not allow the gel to boil.
    3. Keep the bactoagar warm in a water bath at 60 °C.
    4. Keep at 4 °C between experiments.
  2. Embedding of spheroids.
    1. On day 1, for each condition, pool a minimum of 12 spheroids in a 1.5 mL tube.
    2. Wash once with 1 mL of ice-cold 1x PBS.
    3. To fix the spheroids, add 1 mL of 4% paraformaldehyde.
      NOTE: Handling of paraformaldehyde should be performed in a fume hood.
    4. Let them incubate for 24 h at RT.
    5. 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 °C until use.
    6. Wash spheroids twice with 1 mL of ice-cold 1x PBS.
    7. Aspirate most of the 1x PBS (leaving approximately 100 µL at this point is practical for handling the spheroids).
    8. Prepare a 20 µL 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).
    9. Make an agarose gel drop on a microscope slide. Place the slide on a warm heating block to prevent the agarose from solidifying.
    10. Using the modified pipette tip (see 6.2.8), catch as many spheroids as possible in a volume of 15-20 µL.
    11. Carefully inject the 15-20 µL 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.
    12. Let the agarose gel drop harden by incubating for 5-10 min at RT or at 4 °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.
    13. Cover the plastic tissue cassettes in 70% ethanol.
      NOTE: At this point the spheroids can be used directly or stored for months.
    14. Embed the agarose-embedded spheroid in paraffin, section into 2-3 µm thick layer slides and stain with hematoxylin and eosin or subject to immuno-histological staining.

Results

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...

Discussion

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...

Disclosures

The authors declare no conflict of interest.

Acknowledgements

We are grateful to Katrine Franklin Mark and Annette Bartels for excellent technical assistance and to Asbjørn Nøhr-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).

Materials

NameCompanyCatalog NumberComments
2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI)Invitrogen# C10595 For staining nuclei
5-Fluorouracil (5-FU)Sigma-Aldrich#F6627Component in chemotherapeutic treatment
5-(N-ethyl-isopropyl) amiloride (EIPA)Life Technologies#E3111Inhibitor of NHE1
Antibody against PARP and cPARPCell signaling#9542Used in western blotting
Antibody against Ki-67Cell signaling#9449Used for IHC
Antibody against p53Cell Signaling #2524 Used for IHC
Antibody against β-actinSigma A5441Used in western blotting
BactoagarBD Bioscience#214010Used for agarose gel preparation
Benchmark protein ladderInvitrogen#10747-012Used for SDS-PAGE
Bio-Rad DC Protein Assay kitBio-Rad Laboratories#500-0113, #500-0114, #500-0115  Used for protein determination from lysates
Bürker chamberMarienfeld610311For cell counting 
BX63 epifluoresence microscopeOlympusUsed for fluorescent imaging
CellTiter-Glo 3D Cell Viability AssayPromega#G9681Used for the cell viability assay
CisplatinSigma-Aldrich#P4394 Component in chemotherapeutic treatment
Corning Spheroid Microplate, 96 well, Black with clear round bottom,  Ultra-low attachment, With lid, SterileCorning#4520Used for growing spheroids with luminescence measurements as end point
Corning 96 well, clear round bottom,  Ultra-low attachment microplate, With lid, SterileCorning#7007Sufficient for spheroid growth without luminescence measurements as end point
Criterion TGX Precast GelsBio-Rad5671025Used for SDS-PAGE
DoxorubicinAbcam#120629Component in chemotherapeutic treatment
FLUOStar Optima Microplate readerBMG LabtechUsed for recording luminescence 
Formaldehyde VWR Chemicals #9713.1000 Used for cell fixation
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane MatrixGibco#A1413202Keep at 4 °C to prevent solidification. Referred to as rBM in the protocol.
Heat-inactivated FBSSigma#F9665Serum for growth media
ImageJNIHScientific Image analysis
Medim Uni-safe casetteMedim Histotechnologie10-0114Used for storage of embedded spheroids
Mini protease inhibitor cocktail tabletsRoche Diagnostics GmBH # 11836153001Used for lysis buffer preparation
MZ16 microscopeLeicaUsed for light microscopic images
NuPAGE LDS 4x Sample Buffer Invitrogen#NP0007Used for western blotting
Pierce ECL Western blotting substrateThermo scientific#32106Used for western blotting
Ponceau SSigma-Aldrich#P7170-1LUsed for protein band staining
Prism 6.0GraphpadScientific graphing and statistical software
Propidium iodide (1mg/ml solution in water)Invitrogen P3566Light sensitive 
Sterile reservoirs, multichannelSPL lifesciences21002Used for seeding cells for spheroid formation
Superfrost Ultra-Plus Adhesion slide Menzel-Gläser#J3800AMNZMicroscope glass slide used for embedding
TamoxifenSigma-Aldrich#T5648Used as chemotherapeutic treatment
Trans-blot Turbo 0.2 µm nitrocellulose membranesBio-Rad#170-4159Used for western blotting
Tris/Glycine/SDS running buffer Bio-Rad #161 0732Used for SDS-PAGE
Trypsin-EDTA solutionSigma#T4174 Cell dissociation enzyme

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