Assessment of Antitumor and Antiproliferative Efficacy and Detection of Protein-Protein Interactions in Cancer Cells from 3D Tumor Spheroids

Abstract

When compared to two-dimensional (2D) cell cultures, 3D spheroids have been considered suitable in vitro models for drug discovery research and other studies of drug activity. Based on different 3D cell culture procedures, we describe procedures we have used to obtain 3D tumor spheroids by both the hanging-drop and ultra-low-attachment plate methods and to analyze the antiproliferative and antitumor efficacy of different chemotherapeutic agents, including a peptidomimetic. We have applied this method to breast and lung cancer cell lines such as BT-474, MCF-7, A549, and Calu-3. We also describe a proximity ligation assay of the cells from the spheroid model to detect protein-protein interactions of EGFR and HER2.

Basic Protocol 1:

Growth of 3D spheroids using the hanging-drop method

Basic Protocol 2:

Growth of spheroids using ultra-low-attachment plates

Support Protocol 1:

Cell viability assay of tumor spheroids

Support Protocol 2:

Antiproliferative and antitumor study in 3D tumor spheroids

Support Protocol 3:

Proximity ligation assay on cells derived from 3D spheroids

INTRODUCTION

Traditionally, major in vitro cellular assays are carried out using 2D cell culture platforms. Although 2D cell culture is a valuable method in drug discovery, it has some critical limitations. In vivo, almost every cell is surrounded by other cells and a matrix with a unique microenvironment, and 2D cell culture cannot mimic such an environment (Edmondson, Broglie, Adcock, & Yang, 2014; Huh, Hamilton, & Ingber, 2011). On the other hand, cells grown in three-dimensional (3D) culture platforms form aggregates and spheroids (Khaitan, Chandna, Arya, & Dwarakanath, 2006; Kim, 2005). The heterogeneity of cellular populations in found 3D spheroids reflects one of the most characteristic features of in vivo tumors (Baillargeon et al., 2019; Breslin & O’Driscoll, 2013; Costa et al., 2016; Duval et al., 2017; Emerman & Pitelka, 1977; Gurski, Petrelli, Jia, & Farach-Carson, 2010; Jensen & Teng, 2020; Kenny et al., 2007; Lee, Pathak, & Jeong, 2019; Poggi et al., 2021; Roper & Coyle, 2022; Van Zundert, Fortuni, & Rocha, 2020; Vidi, Bissell, & Lelievre, 2013; Zanoni et al., 2016; Zhang & Manninen, 2019; Zhao et al., 2019).

There are reports of 3D cell culture of different cancer cell lines, such as BT-474, SKBR-3, and MCF-7 (Boyer et al., 2021; Han, Kwon, & Kim, 2021; Harma et al., 2010; Meenach et al., 2016; Pulze et al., 2020; Weiswald, Bellet, & Dangles-Marie, 2015; Yakavets et al., 2020). Although 3D spheroid formation has been reported mostly for breast, lung, and colon cancer cells, it has not been widely reported for Calu-3 lung cancer cells because of their slow growth. Calu-3 cell models have, however, been reported in a human airway model of inflammation and in organ-on-a-chip (Camargo, Shamis, Assis, & Mitrani, 2019; Zhu, Chidekel, & Shaffer, 2010) and solid synthetic scaffold applications.

Here, we present two different scaffold-free methods suitable for 3D culture techniques, the first using hanging-drop culture (Polonchuk et al., 2017) and the second using ultra-low-attachment (ULA) multiwell plates (Mittler et al., 2017; Zanotelli et al., 2020), to culture tumor cells. Our procedures are adapted specifically to Calu-3 and A549 lung cancer cells along with two breast cancer cell lines, BT-474 and MCF-7, that overexpress HER2 and express HER2 at a basal level, respectively. Furthermore, we describe a proximity ligation assay (PLA; Fredriksson et al., 2002; Soderberg et al., 2006) that is performed by breaking the spheroids to visualize protein-protein interactions between EGFR and HER2, which are important in lung and breast cancer. As far as we know, there are no previous reports of PLA assays carried out on 3D spheroids. The in vitro PLA can also reveal mechanistic aspects of biochemical pathways that are important for cell signaling in cancer cells. 3D cell culture are an in vitro cell-based system that can mimic in vivo cell features and environment, leading to more predictable results for in vivo preclinical studies. This can facilitate screening compounds in an in vitro assay system before a lead compound is tested in an animal model and thereby help accelerate drug discovery (Edmondson et al., 2014). An overview of the assay and PLA detection is shown in Figure 1.

Figure 1.

An overview of methods for 3D spheroid formation and detection of protein-protein interaction in cells from 3D spheroids.

STRATEGIC PLANNING

Spheroid formation depends on the type of cancer cell line used, and hence cell growth and spheroid formation must be optimized before the antitumor activity of compounds can be evaluated. Some spheroids form aggregates of cells without clear boundaries, and hence a method of volume measurement with an outer boundary needs to be established. We present such a method; further details of spheroid volume measurement have been discussed elsewhere (Roper & Coyle, 2022).

In planning out experiments, the following issues should be taken into consideration. For spheroid growth, different initial seeding densities need to be used for different cell lines because spheroid formation depends on multiple factors such as cell type, duration of the growth phase, and the desired spheroid size. The appropriate seeding density must therefore be determined for each cell line used by initially seeding cells at various densities and assessing their spheroid formation under a microscope.

For cell viability assays, it is important to use spheroids with a starting diameter of 300–500 μm diameter. It is known that spheroids up to 200 μm mimic 3D cell-cell and cell-matrix interactions. Spheroids of cancer cells develop a central secondary necrotic area from starting at a diameter of 500 μm, and larger spheroids may have hypoxic core and be more heterogenous, as cells are at different stages of proliferation. Spheroids >500 μm also may lack sufficient diffusion of oxygen and require the use of a perfusion system. Such spheroids are difficult to handle becausesince they have a secondary necroticsis core. In our assay we used spheroids with a diameter of 300–500 μm that were grown over 72 hr (Amaral, Miranda, Marcato, & Swiech, 2017; Friedrich, Seidel, Ebner, & Kunz-Schughart, 2009; Nath & Devi, 2016; Patra, Peng, Liao, Lee, & Tung, 2016; Zanoni et al., 2016). However, for Calu-3 cells, which do not form the tight spheroids, the initial tumor diameter size may be >500 μm after 72 hr.

Antiproliferative activity (IC₅₀) values of anticancer agents in 2D cells and 3D spheroids may differ, with higher concentrations of compounds usually being required to achieve an IC₅₀ value in the 3D spheroids. The PLA require several washing steps, most of them done in multiwell chamber slides. The PLA should be performed on 2D cell cultures before it is attempted in cells from 3D spheroids. Tumor spheroids can be photographed using a brightfield microscope with a camera attachment. However, to view the fluorescence from PLA probes, a fluorescence microscope with red (for PLA) and blue (for nuclei) fluorescence detection is needed. Slides from PLA assays can be read immediately after the experiment or kept at 4°C and imaged within the next 2–3 days.

NOTE: All cell culture incubations are performed in a 37°C, 5% CO₂ incubator unless otherwise specified.

CAUTION: Cell culture experiments should be performed in a biosafety cabinet.

BASIC PROTOCOL 1

GROWTH OF 3D SPHEROIDS USING THE HANGING-DROP METHOD

This protocol describes the hanging-drop plate method to grow the 3D spheroids. Details are given for the BT-474, MCF-7, A549, and Calu-3 cancer cell lines, with which we have experience, but this can be extended to other cell lines with appropriate changes in conditions.

Materials

• Cell line: BT-474, MCF-7, Calu-3, or A549 (ATCC, cat. no. HTB-20, HTB-22, HTB-55, CCL-185)

• Complete cell culture medium appropriate for cell line: e.g., complete RPMI or complete EMEM (see recipe)

• Trypsin-EDTA (Thermo Fisher Scientific, cat. no. 25300062)

• Biosafety cabinet for cell culture (NUAIR, Class II Type A/B3, Plymouth, MN)

• Corning^® Stripette^® disposable serological pipets, polystyrene, sterile, plugged (VWR, cat. no. 29443-047, 29443-045)

• Bright-Line^™ hemocytometer (MilliporeSigma, cat. no. Z359629)

• Manual cell counter, Tally (VWR, cat. no. 720-1984)

• 15-ml centrifuge tubes (VWR, cat. no. 89174-470)

• Tissue culture flasks (T-25), sterile (Corning, Falcon, cat. no. 29185-302)

• Perfecta3D^® hanging-drop plate (MilliporeSigma, cat. no. HDP1096)

• Eppendorf Research^® Plus Single-Channel Variable Volume Pipettors (VWR, cat. no. 89125-302, 53513-404, 89125-306)

• 1000-μl pipet tips (VWR, cat. no. 83007-386)

• VWR Signature^™ 200 μl pipet tips, graduated (VWR, cat. no. 37001-530)

• Multichannel Pipettes (Thermo Fisher Scientific, cat. no. 4661140N)

• 25-ml disposable polystyrene pipetting reservoirs (VWR, cat. no. 89094-662)

• Shel Lab model SCO2W Benchtop Water Jacketed CO₂ Incubator (Sheldon Manufacturing Inc., cat. no. 1229P57)

• 96-well round-bottom plate with lid, sterile (Corning^®, cat. no. 29442-066)

• 96-well clear-bottom plates (Corning^®, cat. no. 29444-008)

• Micro cover glasses, square, no. 1 (VWR, cat. no. 48366-067)

• Phase-contrast microscope (Olympus America Inc., model no. CK2)

• EVOS XL Core Imaging System (Thermo Fisher Scientific, cat. no. AMEX1000)

• Refrigerated benchtop centrifuge (Jouan, Inc., model no. CR312)

Optimization of spheroid formation

• 1Prepare the cells needed for spheroid formation.

  For cell lines BT-474, MCF-7, Calu-3, and A549 (commercially purchased), our procedure is as followed. Thaw cells stored in vials under liquid nitrogen in a water bath at 37°C for 1–2 min. Once they are thawed, transfer the vials to biosafety hood and transfer the contents of each vial to 9 ml of medium (see Table 1 for appropriate media for each cell) in a 15-ml tube. Centrifuge the tubes for 5–7 min at 466 × g. Visualize the tube and look for cell pellet formation at the bottom of the tube. Take the centrifuge tube to the biosafety hood, remove the clear liquid carefully, and discard. Resuspend the cell pellet in 1 ml complete medium. Place 5 ml complete medium in a T-25 flask, and transfer the 1 ml cell suspension from the 15-ml tube to the T-25 flask. View cells under the microscope to see the floating cells. Place the T-25 flask in the incubator. Passage the cells when the cells reach 70–80% confluency (using trypsin-EDTA to detach the cells from the flask). Use the cell lines after the third passage.

  For spheroid formation, use cell lines within 10 passages.

• 2Count cells using hemocytometer, and then prepare 5-ml suspensions of each type of cells to be used at densities of 125 cells/μl for BT-474, A549, or MCF-7 cells and 250 cells/μl for Calu-3 cells.

  Different initial seeding densities need to be used for different cell lines because spheroid formation depends on multiple factors such as cell type, duration of the growth phase, and the desired spheroid size. This must therefore be determined for each cell line by initially seeding cells at various densities and then visualizing them under a microscope (steps 3–6 below) to check for spheroid formation (Fig. 2).

• 3Seed 40 μl of cell suspension per well into the hanging-drop plate using a multichannel pipet from the top side of the plate.

  Do the manual pipetting very carefully. Insert the pipet tip at least halfway into the well neck region and slowly dispense the cell suspension, ensuring that the hanging drops form on the bottom side of the plate above but not touching the tray. Carefully remove the pipet tips from the well without disturbing the drops.

• 4Check that the hanging drops have formed at the bottom of the well by visual inspection, cover the plate with its lid, and place the assembly in a 37°C, 5% CO₂ incubator.
• 5Evaluate spheroid formation and growth in each drop visually under a light microscope after 8–10 hr.

  After this time, the cells should start to aggregate, leading to the formation of spheroids. By 24 hr after seeding, the formation of tight, very well-defined spheroids should be observed. Assess spheroid formation under the microscope after 24, 48, and 72 hr. BT-474, MCF-7, and A549 cells form well-defined spheroids, whereas Calu-3 forms loosely aggregated spheroids (Fig. 2). Our studies suggest that 5000 cells per well of BT-474, MCF-7, or A549 cells, or 10,000 cells per well of Calu-3, in each case in 40 μl of medium per well in HDP 1096 Perfecta 3D^® 96-well hanging-drop plate (3D Biomatrix) should successfully form spheroids.

• 6Visualize and photograph the spheroids using a digital inverted microscope (EVOS XL Core Imaging System) at different time points 0, 24, 48, and 72 hr (Fig. 2).

Table 1.

Media for Each Cell Line and Harvesting Conditions (when Cells Are Harvested Directly from Original Vial from ATCC)

S.no.	Cell lines	Culture medium	Conditions	Growth rate	Confluency
1.	BT-474	Roswell Park Memorial Institute 1640 medium (RPMI-1640)	37°C, 5% CO2	2–3 days	70%−80%
2.	MCF-7	RPMI-1640	37°C, 5% CO2	3–5 days	70%−80 %
3.	Calu-3	Eagle’s Minimum Essential Medium (EMEM)	37°C, 5% CO2.	5–7 days	70%−80%
4.	A549	RPMI-1640	37°C, 5% CO2	2–3 days	70%−80%

Figure 2.

Spheroids of MCF-7, BT-474, A549, and Calu-3 cell lines obtained in 24 and 72 hr by hanging-drop plating method. Scale bars, 1000 μm.

Transfer of spheroids from hanging-drop plates to 96-well plates for assays

For any antiproliferative activity assay, a hanging-drop plate is not suitable; hence, spheroids need to be transferred to 96-well plates or other type of plates/slides as required for washing. After 72 hr of incubation, visually assess the spheroids and transfer the uniform-appearing spheroids to the 96-well round-bottom plate using the following steps.

• 7Remove the lid and the bottom tray from the 96-well hanging-drop plate, and carefully place the hanging-drop plate containing the spheroids on top of the 96-well round-bottom plate such that the wells of the two plates are aligned with each other.

  A hanging-drop plate assembly has three parts: bottom tray, center hanging-drop holes, and lid. A round-bottom 96-well plate assembly comes with a lid. When the center part of the hanging-drop plate is placed over the round-bottom 96-well plate, the assembly should fit inside, as shown in Figure 3.

• 8After aligning the wells, use 100 μl of serum-free medium to transfer the spheroids: Slowly dispense 100 μl per well of medium onto the wells of the hanging-drop plate to push the spheroids down into the wells of the 96-well round-bottom plate below using a manual micropipettor (Fig. 3C).

  Carefully perform the method to ensure a uniform transfer of the spheroids, without breaking them. Successful transfer of the tightly aggregated MCF-7, BT-474, and A549 spheroids can be easy, but transferring Calu-3 cell spheroids can be quite challenging, as those tend to break up during transfer due to their loose aggregation.

Figure 3.

Transfer of spheroids from hanging-drop plate to 96-well plates. (A) Assembly of hanging-drop plate showing the lid, the center part of the hanging-drop plate, and the tray. (B) The center part of the hanging-drop plate is placed aligned over 96 wells. (C) Assembly of hanging-drop plate placed over 96-well plate. Spheroids from hanging-drop plate to wells of a 96-well plate using a pipet.

BASIC PROTOCOL 2

GROWTH OF SPHEROIDS USING ULTRA-LOW-ATTACHMENT PLATES

This protocol describes the use of ultra-low-attachment (ULA) plates to grow 3D spheroids from the BT-474, MCF-7, A549, and Calu-3 cancer cell lines.

Materials

• Cell line: BT-474, MCF-7, Calu-3, or A549 (ATCC, cat. no. HTB-20, HTB-22, HTB-55, CCL-185)

• Complete cell culture medium (see recipe; i.e., complete RPMI or complete EMEM)

• 96-well Corning^® spheroid microplates (MilliporeSigma, cat. no. CLS4515)

• Corning^® Stripette^® disposable serological pipets, polystyrene, sterile, plugged (VWR, cat. no. 29443–047, 29443–045)

• Eppendorf Research^® Plus Single-Channel Variable Volume Pipettors (VWR, cat. no. 89125–302, 53513–404, 89125–306)

• 1000-μl pipet tips (VWR, cat. no. 83007–386)

• VWR Signature^™ 200-μl pipet tips, graduated (VWR, cat. no. 37001-530)

• Multichannel Pipettes (Thermo Fisher Scientific, cat. no. 4661140N)

• 25-ml disposable polystyrene pipetting reservoirs (VWR, cat. no. 89094-662)

Spheroid formation protocol

1. Use Corning^® 96-well spheroid microplates for spheroid growth. Harvest all cells using the standard method and obtain cell suspension (Basic Protocol 1, step 1). Count the cells, and prepare 5 ml of cell suspension at a density of 50 cells/μl for BT-474, 20 cells/μl for A549 cells, 50 cells/μl for MCF-7 cells, or 100 cells/μl for Calu-3 cells.

2. Seed 100 μl cell suspension per well manually into the Corning^® 96-well spheroid microplates using a multichannel pipet from the top side of the plate. Perform manual pipetting very carefully. Carefully remove the pipet tips from the well without disturbing the drop.

3. Place the plate in the incubator and incubate at 37°C, 5% CO₂.

4. Evaluate spheroid formation and growth visually after 24, 48, and 72 hr using a light microscope. The cells should visibly start to aggregate, leading to spheroid formation (this usually takes 1–3 hr after seeding).

   The time and nature of spheroid formation are dependent on cell type and cell densities. BT-474, MCF-7, and A549 form tight, very well-defined spheroids by 24 hr after seeding, whereas Calu-3 forms loosely aggregated spheroids (Fig. 4).

   With this method, there is no requirement to transfer the spheroids. Other experiments requiring multiwell plates can be performed using the same Corning^® 96-well spheroid microplates.

   Spheroids are not stored; usually, they are processed immediately for further studies, such as antiproliferative and antitumor studies.

Figure 4.

Results of ultra-low-attachment plating method: images of spheroids made from the MCF-7, BT-474, A549, and Calu-3 cell lines after 24 and 72 hr. Scale bars, 1000 μm.

SUPPORT PROTOCOL 1

CELL VIABILITY ASSAY OF TUMOR SPHEROIDS

This section describes a procedure for cell viability assays of tumor spheroids prepared using either the hanging-drop or ultra-low-attachment plate method, which is carried out to evaluate the viability of the cells during spheroid formation. Spheroid formation and growth are monitored using the commercial CellTiter-Glo^™ 3D Cell Viability Assay (Promega).

Materials

• Spheroids from Basic Protocol 1 (step 6; hanging drop method) or Basic Protocol 2 (step 4; ultra-low-attachment plate method)

• Promega CellTiter-Glo^™ 3D Cell Viability Assay (Promega Corporation, cat. no. PRG9683)

• Corning^® Stripette^® disposable serological pipets, polystyrene, sterile, plugged (VWR, cat. no. 29443-047, 29443-045)

• Eppendorf Research^® Plus Single-Channel Variable Volume Pipettors (VWR, cat. no. 89125-302, 53513-404, 89125-306)

• 1000-μl pipet tips (VWR, cat. no. 83007-386)

• VWR Signature^™ 200-μl pipet tips, graduated (VWR, cat. no. 37001-530)

• Perfecta3D^® hanging-drop plate (MilliporeSigma, cat. no. HDP1096)

• Corning^® spheroid microplates (MilliporeSigma, cat. no. CLS4515)

• 96-well plate reader

• 96-well sterile round-bottom plate with lid (Corning^®, cat. no. 29442-066)

• 15-ml centrifuge tubes (VWR, cat. no. 89174-470)

• Tissue culture flasks, sterile (Corning, Falcon, cat. no. 29185-302)

• Multichannel Pipettes (Thermo Fisher Scientific, cat. no. 4661140N)

• 25-ml disposable polystyrene pipetting reservoirs (VWR, cat. no. 89094-662)

• Micro cover glasses, square, no. 1 (VWR, cat. no. 48366-067)

• 1.Thaw Promega CellTiter-Glo 3D Cell Viability Assay reagent and allow it to come to room temperature.
• 2a.Hanging-drop method: At different time points (24, 48, and 72 hr), transfer spheroids (from Basic Protocol 1, step 6) to a 96-well round-bottom plate as described in Basic Protocol 1, steps 7 and 8, and add 100 μl CellTiter-Glo 3D cell viability assay reagent to each well.

  It is necessary to establish a growth curve for each cell line before performing the antiproliferative assay by evaluating their growth at different time points.

• 2b.Ultra-low-attachment plate method: Add 100 μl CellTiter-Glo 3D reagent to each well of the 96-well ultra-low-attachment plate containing spheroids and culture medium.
• 3.Vigorously mix the contents of the wells by pipetting or by using a shaker for 5 min to induce cell lysis.
• 4.Incubate the plate for an additional 20 min to stabilize the luminescence signal.
• 5.Record luminescence using a 96-well plate reader. Use wells containing medium only as controls.
• 6.Plot a graph of relative luminescence with respect to time to assess cell viability.
• 7.Compare the cell viability of the spheroids for the different cell lines at 0, 24, 48, and 72 hr as indicated by the luminescence data (Figs. 5 and 6).

Figure 5.

Cell viability assay graph of A549, Calu-3, BT-474, and MCF-7 cell lines obtained by hanging-drop plating method.

Figure 6.

Cell viability assay graph of A549, Calu-3, BT-474, and MCF-7 cell lines obtained by ultra-low-attachment plating method.

SUPPORT PROTOCOL 2

ANTIPROLIFERATIVE AND ANTITUMOR STUDIES IN 3D TUMOR SPHEROIDS

3D spheroids are a superior model for tumor studies compared to 2D cultures. Here we describe a protocol to evaluate the antiproliferative and antitumor effects of chemotherapeutic and targeted therapy agents as well as a peptidomimetic (doxorubicin, lapatinib, and peptide SFTI-G5, respectively) using different tumor spheroids of BT-474, MCF7, A549, or Calu-3 through a cell viability assay and measurement of spheroid sizes in different treatment groups.

The antiproliferative effect of compounds on tumor spheroid was determined by using the CellTiter-Glo Luminescent Cell Viability Assay (Vinci et al., 2012). This assay measures the amount of ATP produced to determine cell viability.

Materials

• Spheroids from Basic Protocol 1 (step 6; hanging drop method) or Basic Protocol 2 (step 4; ultra-low-attachment plate method)

• Complete cell culture medium: e.g., complete RPMI or complete EMEM (see recipe)

• Compounds of interest:

  • Doxorubicin HCl (Astatic cat. no. 30760)
  • Lapatinib (Sigma Aldrich cat. no. SML2259–50MG)
  • SFTI-G5 (synthesized in the laboratory by solid-phase peptide synthesis; Singh et al., 2021)

• DMSO

• Promega CellTiter-Glo^™ 3D Cell Viability Assay (Promega Corporation, cat. no. PRG9683)

• Micro cover glasses, square, no. 1 (VWR, cat. no. 48366–067)

• Eppendorf Research^® Plus single-channel variable-volume pipettors (VWR, cat. no. 89125-302, 53513-404, 89125-306)

• 1000-μl pipet tips (VWR, cat. no. 83007-386)

• VWR Signature^™ 200-μl pipet tips, graduated (VWR, cat. no. 37001-530).

• Corning^® Stripette^® disposable polystyrene serological pipets, sterile, plugged (VWR, cat. no. 29443-047, 29443-045)

• 96-well round-bottom plate with lid, sterile (Corning^®, cat. no. 29442-066)

• Perfecta3D^® hanging-drop plate (MilliporeSigma, cat. no. HDP1096)

• Corning^® spheroid microplates (MilliporeSigma, cat. no. CLS4515)

• Plate reader with a luminescence detection option.

Antiproliferative assay

1. To study the antiproliferative activity of the compounds, use tumor spheroids of uniform size after 72 hr of growth. A visual inspection of spheroids under the microscope can be helpful for selecting uniform-sized tumor spheroids. For the cell lines BT-474, MCF-7, and A549, intact, uniform spheroids should be formed by 72 hr; the diameter we measured was 300–500 μm. For Calu-3 cells, which form loose spheroids, the diameter measured was >500 μm. Overall, the 72-hr time point can be considered as the optimum time for uniform spheroid formation for these four cell lines.

2. Prepare doxorubicin, lapatinib, and SFTI-G5 at 5 and 10 μM each in serum-free medium. First, dissolve a known amount of compound in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution, and then prepare the working solutions by diluting them with serum-free medium make sure that the DMSO concentration is <1% in working solution (now onwards called compounds/drugs).

   Doxorubicin and lapatinib were used as positive controls and the spheroid-treated serum-free medium alone as a negative control. Doxorubicin is a potent cytotoxic agent (Minotti, Menna, Salvatorelli, Cairo, & Gianni, 2004) and makes a good positive control because it can kill cancer cells and shrink the tumor spheroids. Lapatinib is a tyrosine kinase inhibitor that inhibits the phosphorylation of the tyrosine kinase domain in EGFR family proteins (Diaz et al., 2010), suppressing growth signaling in tumor cells and resulting in their apoptosis. In our study, lapatinib served as positive control in HER2-positive cells such as BT-474, A549, and Calu-3 cells. SFTI-G5 is a protein-protein interaction inhibitor of EGFR dimers (Singh et al., 2021).

   If the IC₅₀ value of the compound in 2D cell culture is known, one can prepare the dilution of compounds/drugs based on IC₅₀ values. 3D spheroids need a higher concentration of drugs compared to 2D cell culture models to observe the same effect.

3. For spheroids generated by the hanging-drop method (Basic Protocol 1) only: Transfer the spheroids to 96-well round-bottom plate as described in Basic Protocol 1, steps 7 and 8, using 100 μl serum-free medium containing different amounts of compounds/drugs of interest. Incubate for 72 hr at 37°C, 5% CO₂.

   With the ultra-low-attachment plate method, spheroids need not be transferred to 96-well plates; instead, continue the experiments in the ultra-low-attachment plates.

4. After 72 hr, add 100 μl CellTiter-Glo^® Reagent.

5. Mix the contents of the well vigorously for 5 min. Incubate for 20 min at 37°C, 5% CO₂.

6. Read the plate using a plate reader with a luminescence detection option.

7. Compare the relative cell viability at two different concentrations of the compound with respect to the control (5 μM and 10 μM).

   We compared 3D tumor spheroids from four different cell lines, A549, Calu-3, MCF-7, and BT-474 (Figs. 7 and 8).

Figure 7.

Graphs of antiproliferative activity of lapatinib, doxorubicin, and SFTI-G5 at 5 and 10 μM concentrations on A549 and Calu-3 spheroids.

Figure 8.

Graphs of antiproliferative activity of lapatinib, doxorubicin, and SFTI-G5 at 5 and 10 μM concentrations on BT-474 and MCF-7 spheroids.

Antitumor studies in 3D spheroids

We have performed this study using HER2-positive BT-474 spheroids and HER2-negative MCF-7 tumor spheroids.

• 8.Prepare compounds (doxorubicin, lapatinib, and SFTI-G5) at 1, 5, and 10 μM as described in step 2 above.
• 9a.Hanging-drop method: After 72 hr of growth, transfer the spheroids of uniform size (see step 1) to a 96-well round-bottom plate as described in Basic Protocol 1, steps 7 and 8, using 100 μl serum-free medium containing the desired concentrations of the different compounds being tested (or plain serum-free medium for controls).
• 9b.Ultra-low-attachment plate method: After 72 hr of incubation (Basic Protocol 2, step 4), check that the spheroids are of uniform size and add the compound in serum-free medium to the existing medium using a micropipet.

  From this point on, the protocol is the same for both types of spheroids. Compound is added to the existing medium without removing any medium.

• 10.Image the plates to obtain a day 0 image for later comparison.
• 11.Incubate the plates at 37°C, 5% CO₂. Every other day till day 5 of treatment, replenish the compounds in the medium by adding 15 μl of serum-free medium containing 10 μM of the appropriate compound (or plain serum-free medium for controls).
• 12.Image the spheroids every day up to day 5. Determine the antitumor efficacy of the compounds by comparing the relative volume increases in the treatment groups versus the control group from day 0 to day 5.

  For the hanging-drop method, see Figure 9 (BT-474, MCF-7) for sample results. For the ultra-low-attachment-plate method, see Figure 9 (A549 and Calu-3).

• 13.Estimate the dimensions of the spheroid using ImageJ software by measuring the longest and shortest diameters (L and W, respectively).
• 14.Calculate the approximated volume (V) of each spheroid, estimated as (W² × L)/2.
• 15.Plot a graph of 3D spheroid volume with respect to time. Analyze the statistical significance of the results (Figs. 10 and 11).

Figure 9.

Representative images of tumor spheroids obtained from BT-474, MCF-7, A549, and Calu-3 cells. Control without treatment and spheroids after treatments on day 4 are shown; notice the disaggregation of spheroids after treatment with lapatinib or compound SFTI-G5. For such spheroids, tumor volume is measured by length and breadth at the outer edge of the spheroids. Scale bars, 1000 μm.

Figure 10.

Antitumor activity of compounds. Activity in (A) A549 and (B) Calu-3 tumor spheroids (ultra-low-attachment plate) is shown. Dose-response curves are plotted for different compounds. Data are presented as mean ± SD (n = 3). ***p < 0.001, **p < 0.01, and *p < 0.05 compared with control.

Figure 11.

Antitumor activity of the compounds. Activity in (A) BT-474 and (B) MCF-7 tumor spheroids (hanging-drop plate) is shown. Data are presented as mean ± SD (n = 3). ***p < 0.001 and *p < 0.05 compared with control.

SUPPORT PROTOCOL 3

PROXIMITY LIGATION ASSAY ON CELLS DERIVED FROM 3D SPHEROIDS

Protein-protein interactions (PPI) in cells and tissues can be visualized in a cell through a PLA. Primary and secondary antibodies can be directed against protein pairs that are <40 nm apart and can be detected using DNA fluorescent probes (Fredriksson et al., 2002; Soderberg et al., 2006). In this particular case, EGFR:HER2 dimers are present in HER2-overexpressing breast and lung cancer cell lines, and the interaction of EGFR and HER2 can be visualized by using PLA in the form of red fluorescence dots. A higher number of red fluorescence dots indicates more PPI and a lower number indicates less PPI. Our main aim in the PLA experiment was to evaluate whether the PPI can be visualized in the 3D spheroids in this fashion. Such methods are useful for drug screening to evaluate whether compounds of interest inhibit PPI in 3D spheroids similarly to in the tumor environment. This assay was carried out on A549 lung cancer cells. A549 lung cancer cells overexpress the HER2 protein and have KRAS mutation (Bunn et al., 2001; Suzawa et al., 2016).

Materials

• Spheroids prepared from cell line A549 (ATCC, cat. no. CCL-185) according to Basic Protocol 1, steps 1–6

• Serum-free medium: e.g., RPMI

• Complete cell culture medium: e.g., complete RPMI (see recipe)

• Primary antibodies for binding to specific proteins

  • EGFR ANTIBODY (ENZO, CAT. NO. ADI-CSA-330-E)
  • HER-2 ANTIBODY (ENZO, CAT. NO. ALX-810–227-L001)

• Duolink in situ red mouse/rabbit PLA kits (Sigma Aldrich, cat. nos. DUO 92004, DUO 92002)

• Methanol, ice cold

• Duolink in-situ detection Reagent Red (Sigma Aldrich, cat. no. 92008)

• Therapeutic agent of interest: e.g., SFTI-G5 (synthesized in the laboratory by solid-phase peptide synthesis; Singh et al., 2021)

• Positive control agent: e.g., pertuzumab (provided by Genentech Inc. under a Materials Transfer Agreement)

• Nail polish (optional)

• Nunc^® Lab-Tek^® 8 well Chamber Slide^™ system (Sigma Aldrich, cat. no. C7182)

• Humid chamber (e.g., prepared by using a pipet box and wet paper towels; Fig. 12)

Figure 12.

Preparation of humid chamber from pipet box.

Preparation and drug treatment of 3D spheroids

• 1At 72 hr after cell seeding (Basic Protocol 1, step 6), transfer the A549 3D spheroids to wells of a 96-well round-bottom plate as described in Basic Protocol 1, steps 7 and 8, using 100 μl serum-free medium.
• 2Add 100 μl serum-free medium to the control well.
• 3Add 100 μl of 10 μM SFTI-G5 (prepared as described in Support Protocol 2, step 2).
• 4Add 100 μl of 1 μM pertuzumab (from stock solution) in serum-free medium as a positive control.
• 5Incubate the spheroids in the plate for 24 hr (95% air, 5% CO2).

Proximity ligation assay

Coating of spheroids in eight-well chamber slides

• 6After incubation with SFTI-G5 or pertuzumab or without any treatment (just in serum-free medium) for 24 hr, break up the spheroids in their wells by vigorous pipetting and transfer the broken spheroids from the 96-well plate to eight-well chamber slides.
• 7Add 100 μl serum-free medium to cells in chamber slides using a pipet and incubate for 6–8 hr to allow further of attachment of cells.

Blocking and fixation

• 8Wash the broken spheroids on the eight-well chamber slides with wash buffer A (provided in the Duolink kit).
• 9Remove the wash buffer using a micropipet, add 100 μl ice-cold methanol per chamber using a micropipet, and incubate slides for 15 min at −20°C to fix the broken spheroids.
• 10Remove methanol using a micropipet and wash the fixed cells with 100 μl wash buffer A per chamber, and then gently remove the wash buffer using the micropipet. Gently add 100 μl blocking solution per chamber using the micropipet and incubated 90 min at room temperature.

Primary antibody addition

• 11Prepare a 2:1 cocktail of EGFR and HER2 antibodies in 5% BSA. Add 100 μl of the antibody cocktail to each plate chamber, and incubate slides overnight in a humid chamber by gentle shaking.

Addition of probes

• 12Wash the wells with wash buffer A.
• 13Prepare the two probe solutions in 2% BSA solution at a 1:1:4 (v/v/v) ratio of positive probe/negative probe/blocking solution.
• 14Add of 60 μl of this solution to each well, and incubate plates 1 hr at 37°C in a humid chamber.

Addition of ligase solution

• 15Wash each well with 100 μl wash buffer A twice.
• 16Prepare the ligase solution following the manufacturer’s protocol.

  This consists of a 1:8:31 (v/v/v) ratio of the ligase enzyme/ligation buffer/ultrapure water.

• 17Add 40 μl of the ligase solution to each well and incubate 30 min at 37°C.

Addition of amplification and polymerase solution

• 18Wash each well with wash buffer A twice.
• 19Prepare DNA polymerase/amplification solution following the manufacturer’s protocols.

  The mixture consists of 0.5:8:31.5 (v/v/v) 0.5:8:31.5 DNA polymerase/amplification red/ultrapure water. Add 40 μl of the prepared polymerase/amplification solution to each chamber and incubate 100 min in a humid chamber in the dark.

Final washing

• 20After incubation, wash the wells with wash buffer B (provided in the Duolink kit) for 10 min.
• 21Remove the walls of chambers.
• 22Add 10–20 μl of DAPI with mounting medium onto the slides and cover with a coverslip.

  Nail polish can be used to seal the edges of the coverslip. This will help prevent movement of cover slip during imaging. Make sure that the nail polish is dried before using the slides for imaging, as if not dry, it can be transferred to microscope parts.

Imaging

• 23View the slides under a fluorescence microscope.

  For imaging, we use a Olympus BX63 microscope fitted with deconvolution optics using DAPI, FITC, and Texas Red filters and 100× magnification. The slides can be kept at 4°C in the dark, and microscopy can be done for up to 2–3 days.

• 24Process the images as appropriate (we use CellSens dimension software). Red dots indicate the protein-protein interaction (Fig. 13).

  The antibody pertuzumab is used as a positive control for comparing PPI inhibition and wells without any treatment as a negative control.

Figure 13.

PLA of 3D spheroids on A549 cells overexpressing HER2. Red fluorescence dots indicate the EGFR:HER-2 interaction. Control spheroids show the greatest number of red dots compared to SFTI-G5 and pertuzumab treated, indicating inhibition of EGFR:HER-2 interaction. Expanded regions are shown for PPI (red dots in control) and its inhibition by SFTI-G5 and pertuzumab. Scale bars, 10 μm.

REAGENTS AND SOLUTIONS

Complete cell culture media

Carry out all preparation steps in a biosafety hood.

Upon purchase, thaw penicillin/streptomycin (ATCC, cat. no. 30–2300), divide into 5.5-ml aliquots in 15-ml Falcon tubes, and store at −20°C. Similarly, thaw FBS (ATCC, cat. no. 30–2021), filter through Nalgene filter (Thermo Fischer scientific, cat. no. 156–4020), divide into 50-ml aliquots in Falcon tubes, and store at −20°C.

Complete RPMI medium

1. Transfer RPMI (incomplete medium; ATCC, cat. no. 30–2001) from refrigerator to a room-temperature water bath and let sit for ~20 min to bring it to room temperature.

2. Meanwhile, allow a 5.5-ml aliquot of penicillin/streptomycin and a 50-ml aliquot of FBS to thaw completely.

3. Add 50 ml FBS to medium and gently mix. Add 5.5 ml penicillin/streptomycin and mix. Finally, add 500 μl insulin solution (10 mg/mL insulin dissolved in 25 mM HEPES, pH 8.2; Sigma Aldrich, cat. no. I0516).

4. Store the complete medium in the refrigerator (2–8°C); the complete medium can be used until the expiration date of the incomplete medium.

Complete EMEM medium

Follow the same procedure as for preparation of complete medium, but starting with EMEM (ATCC, cat. no. 30–2003) as the incomplete medium. Store in the refrigerator (2–8°C); the complete medium can be used until the expiration date of the incomplete medium.

COMMENTARY

Background Information

The 2D cell culture platform is extensively used in cell-based research for in vitro studies, as 2D flat monolayers of cells are very convenient to use and cost effective. However, the use of 2D cell culture for drug discovery purposes, particularly in cancer research, poses several limitations. Cancer tumors are 3D, and 2D cell culture methods do not provide the correct microenvironment, as cells in their normal environments are surrounded by other cells and extracellular matrix in 3D. Cytotoxicity or antiproliferative activity assays based on 2D cell culture for anticancer compounds may result in high rates of failure of drug discovery for anticancer agents (Edwards et al., 2015; Sams-Dodd, 2005). The 3D growth of commercially available cell lines or primary cell culture is considered to provide a more accurate and representative model when screening drugs in vitro to identify cancer-targeting agents. By comparison, 3D cell cultures better represent the microenvironment of tumors such as cell-cell interaction, hypoxia, drug penetration, protein expression, response, and resistance (Padmalayam & Suto, 2012; Zanoni et al., 2016). Unlike in flat 2D cell cultures that are exposed to medium and cell culture dish, in 3D cultures cells are exposed to other cells or to the ECM, a situation closer to their usual physiological state. Cells from 2D cultures may change phenotype and become different from the tissues they were derived from. Thus, methods for 3D culture of cells have been investigated at length for drug discovery and other in vitro research. The idea of 3D cell culture is not new, having been conceived as early as 1907 by Harrison, who grew neural tube fragments from frog embryos in “hanging-drop culture” (Harrison, 1906). The method was reportedly used even earlier by Koch et al. to grow anthrax bacilli (Alhaque, Themis, & Rashidi, 2018). Later the possibility of 3D culture of cancer cells was reported by Petersen, Rønnov-Jessen, Howlett, and Bissell (1992). In the early 1970, Robert Sutherland’s group reported 3D structures formed by Chinese hamster lung cells and coined the word “spheroids” for such formations (Inch, Credie, & Sutherland, 1970). Later, 3D culture methods were further developed and commercial plates are now available for growth of 3D spheroids. Notably, there are differences between 2D and 3D cell cultures in terms of gene expression, cellular morphology, and drug sensitivity. In particular, 2D cultures are more sensitive to drugs than 3D cell cultures (Tung et al., 2011; Weigelt, Lo, Park, Gray, & Bissell, 2010).

There are two types of 3D cell culture techniques: scaffold based and scaffold free. Scaffold-based methods use synthetic or naturally derived polymer support as matrix for cell growth. Although scaffold platforms provide some advantages for cell growth, currently available scaffolds have limitations in terms of obtaining a controlled matrix for cellular physiological growth and mimicking in vivo conditions. Cancer tumor spheroids are often grown using scaffold-free method, and hence we describe those here. Although scaffold-free methods use cell culture medium, some reports indicate that cells within the spheroids produce ECM proteins that fill the intercellular space (Nederman, Norling, Glimelius, Carlsson, & Brunk, 1984). Thus, scaffold-free methods represent a more natural, in-vivo-like condition in which cells self-assemble, grow, and produce ECM proteins. However, depending on the need, one can use any of the methods detailed here for 3D cell culture.

Scaffold-free methods use spheroid formation in suspension using the forced floating method, hanging-drop methods, or agitation-based methods, which rely on self-aggregation of cells. Here we describe the two most widely used approaches, the hanging-drop and ultra-low-attachment plate methods. Hanging-drop microplates are specialized plates with open, bottomless wells that facilitate the formation of a droplet of the cell medium. The droplet allows the cells to form aggregates so as to form spheroids, without attaching to the plate surface, and these are supposed to be a good representation of the cells in the vicinity of a capillary in tumor. Ultra-low-attachment coating, or low-adhesion, plates are created with plate bottoms to which cell have difficulty attaching, and hence promote aggregation of cells. The plates are made with polystyrene and treated with coatings that reduce cell attachment (Friedrich et al., 2009; Ivascu & Kubbies, 2006). In terms of experimental procedures, ultra-low-attachment plates have advantages for manipulation of spheroids as they hold a higher volume of medium than hanging droplets. Another advantage is that the spheroids can remain in the same plate for later experimental procedures. In contrast, spheroids in hanging drops must be transferred to multiwell plate for further analysis, because it is not possible to perform most experiments in hanging drops because the droplet volume is only ~50 μl, and adding testing medium to such a small volume without disturbing the droplet can be difficult. Hanging-drop plates can be used in high-throughput screening, but this requires expertise. On the other hand, ultra-low-attachment plate surfaces carry the complication that they must be uniform during modification and manufacturing in order to function. Any inconsistency of the prepared surface will result in attachment of some cells, affecting spheroid formation. Cells in hanging drops, in contrast, do not touch any polystyrene surfaces and hence aggregate naturally. However, because of forced floating in the ultra-low-attachment method, aggregates can form an almost spherical shape, whereas with the hanging-drop methods, ellipsoids may form, depending on the cell type. Comparison of these methods for different cell lines have been reported (Amaral et al., 2017; Bresciani et al., 2019). Raghavan et al. (2016) have shown that gentle rotation aids the aggregation of cells in both hanging-drop and ultra-low attachment plate methods. Because each method has some limitations (see Troubleshooting), it is suggested that 3D cellular assays be performed using at least two methods, depending on the cell type, during drug discovery.

Protein-protein interactions were traditionally detected through co-immunoprecipitation (Hall, 2005), pull-down assays (Nguyen & Goodrich, 2006), yeast two-hybrid assays (Guo, Rajamaki, & Valkonen, 2008), or fluorescence resonance energy transfer (FRET) or bioluminescence resonance transfer (BRET) methods using cell culture (Karpova & McNally, 2006; Kocan & Pfleger, 2011). Co-immunoprecipitation methods use lysis of cells and then detection of proteins using capturing antibodies, whereas FRET/BRET methods involve modification of proteins in cells using donors and acceptors. There was no method to directly detect the proximity of two proteins in cells and tissues without performing considerable manipulation or subjecting the samples to lysis to extract the proteins. The proximity ligation assay (PLA) was reported by Fredriksson et al. (2002) and later commercialized by Olink Biosciences. The assay is useful to detect proteins that are in proximity in situ (at distances <40 nm) at endogenous protein levels and can be applied to in vitro and in vivo samples (Fichter et al., 2014; Roussis, Guille, Myers, & Scarlett, 2016; Soderberg et al., 2006; Trifilieff et al., 2011). Application of PLA to 2D cell culture has been extensively reported in the literature, along with protocols (Alam, 2018; Hegazy et al., 2020). To the best of our knowledge, however, PLA on 3D spheroid cancer cells has not been reported to date. PLA on 3D spheroids could aid drug screening of potential protein-protein interaction inhibitors. As mentioned before, gene and protein expression as well as cell-cell interaction are different in 2D cell culture compared to 3D cell culture. Hence a method to detect protein-protein interaction using 3D cells is valuable for evaluating the expression of proteins in cells that interact in cancer cells. This is particularly important because different cancer cells overexpress different proteins and development of resistance to different cancer treatments changes the expression of proteins due to mutation. In addition to this cell surface expression and interaction of proteins in cancer cells is affected by altered cellular trafficking of these proteins (Sorkin & Goh, 2009; Tomas, Futter, & Eden, 2014). Thus, evaluation of protein-protein interaction to understand the molecular mechanism and resistance is important. The assay can be used as a screening method to look at the trafficking, cell surface expression, and dimerization of proteins.

Troubleshooting

Table 2 lists categories of problems that may arise with this procedure along with their possible causes and solutions.

Table 2.

Critical Parameters and Troubleshooting

Observation	Cause	Resolution
Not all cells form perfect spheroids	Some cells, such as A549, form spherical shape that can be measured with better accuracy, whereas Calu-3 cells form aggregates of cells.	The measurement of diameters must be optimized for these cells. Ultra-low-attachment plates serve better for cells that form aggregates.
PLA could not be performed on intact spheroids, and the red fluorescence dots could not be visualized using the intact spheroids	Fluorescence light emission from deep within the spheroid did not reach the detector. Poor permeability of antibodies and probes into the intact spheroids may also be a factor.	The spheroids were broken up with vigorous pipetting before PLA.
The direct transfer of intact spheroids to 8-well chamber slides for treatment, and further incubation, led to the growth of the spheroids as monolayer cells	When transferred to the chambers, the spheroids started growing as a 2D cell monolayer because of the completely flat surface of the chamber.	Spheroids were transferred to the bottoms of the wells of 96-well plates for treatment. The assay was carried out on broken spheroids.

Understanding Results

The HER-2-positive breast and non-small-cell lung cancer cell lines BT-474, Calu-3, and A549 overexpress HER2, representing aggressive forms of breast and lung cancer (Lee-Hoeflich et al., 2008; Mar, Vredenburgh, & Wasser, 2015; Naik et al., 2021; Takezawa et al., 2012; Wang et al., 2006). The compound SFTI-G5 is a cyclic peptidomimetic that is known to target HER2-overexpressing cancer cells and inhibit the dimerization of EGFR:HER2 and HER2:HER3 (Singh et al., 2021). This compound also inhibits lung tumor growth in an animal model. Antiproliferative activity of SFTI-G5 was compared with that of lapatinib, a well-known kinase inhibitor of EGFR and HER2 (Diaz et al., 2010). Thus, compounds we call for in these protocols to inhibit cancer cell growth are specific for EGFR and HER2 proteins that are present on the cells we used for the study. MCF-7 is a breast cancer cell line known to have basal-level HER2 expression, and the compound SFTI-G5 does not affect the antiproliferative activity of MCF-7. MCF-7 is used as a negative control. Doxorubicin (Dox), a chemotherapeutic agent known to kill most of cells, is used as another control (Minotti et al., 2004). We determined the antiproliferative activity on 3D tumor spheroids of SFTI-G5 along with lapatinib and doxorubicin at different concentrations and found that doxorubicin at different concentrations exhibited significant antiproliferative activity on tumor spheroids of all cell lines (Figs. 7 and 8). Lapatinib exhibited significant antiproliferative in HER2-positive cell lines but did not significantly affect MCF-7 3D spheroids. Similarly, the compound SFTI-G5 exhibited specificity in targeting HER2-positive tumor spheroids but showed minimal effect on MCF-7 spheroids (Fig. 8B).

Antitumor activity of the compounds was determined by assessing the spheroids’ volumes, calculated from images of the spheroids taken from day 1 to day 5 (Sonju et al., 2022). Representative images of 3D spheroids on day 4 after treatment and control are shown in Figure 9. The treatment groups, including lapatinib, doxorubicin, and SFTI-G5, showed a significant decrease in BT-474 tumor spheroid volume on day 5. Similarly, in MCF-7 tumor spheroids, doxorubicin showed high antitumor activity, but SFTI-G5 had minimal activity. MCF-7 cells have minimal HER-2 expression, while SFTI-G5 is designed to target HER2 domain IV, and hence it has an insignificant effect on MCF-7 tumor spheroids (Figs. 10 and 11).

PLA on cells from 3D spheroids

Protein-protein interactions in cells and tissues can be visualized within a cell through PLAs. Primary and secondary antibodies can be directed against protein pairs that are <40 nm apart and can be detected using fluorescent DNA probes (Fredriksson et al., 2002; Soderberg et al., 2006). In this particular case, EGFR:HER2 dimers are present in HER2-overexpressing breast and lung cancer cell lines, and the interaction of EGFR and HER2 can be visualized by PLA in the form of red fluorescence dots. A higher number of red fluorescence dots indicates more PPI, whereas a lower number of dots indicates lower PPI. Our main aim in the PLA experiment was to evaluate whether PPI can be visualized in the 3D spheroids using PLA techniques. Such methods are useful for drug screening to evaluate compounds designed inhibit PPI in a 3D spheroid similar to the tumor environment. Our designed peptidomimetic SFTI-G5 binds to domain IV of HER2 and inhibits HER2 heterodimerization. Pertuzumab is an antibody to HER2 that is known to bind to HER2 and inhibit EGFR:HER2 dimerization and are used therapeutically to treat breast and lung cancers that overexpress HER2 (Barthelemy, Leblanc, Goldbarg, Wendling, & Kurtz, 2014; Sliwkowski & Mellman, 2013). Pertuzumab is used as a positive control that inhibits HER-2 heterodimerization (Franklin et al., 2004). 3D spheroids that were not treated with compound or antibody-drug exhibited a large number of red fluorescence dots indicative of EGFR:HER2 dimerization. Spheroids treated with the peptide SFTI-G5 or the antibody pertuzumab showed reduced PPI, indicating the inhibition of HER-2 heterodimerization (Fig. 13).

The described 3D spheroid model can be used to elucidate the molecular mechanism of dimerization of proteins as well as in drug screening. 3D tumor spheroids closely resemble solid tumors in vivo in their architecture, physiological responses, gene expression patterns, and drug resistance mechanisms (Amaral et al., 2017; Costa et al., 2016). 3D cell culture is an in vitro cell-based system that can mimic in vivo cell features and environment, providing more predictable results for in vivo preclinical studies. This can help reduce the likelihood of drug failure at later stages of drug discovery and development (Edmondson et al., 2014).

Data Analysis

The spheroid dimensions are measured using ImageJ by measuring the longest and shortest diameters (length [L] and width [W], respectively). Spheroid volume is measured from the diameter of the outer boundaries of the diffuse aggregates of Calu-3 cells. The volume (V) of each spheroid is calculated using the equation (W² × L)/2. Triplicate experiments should be performed and the data transferred to Microsoft Excel and GraphPad Prism (v.8) to perform statistical analysis. Data are represented as mean ± standard deviation. In our particular case, for the antitumor activity of the compounds in 3D spheroids, data were compared to the control, and significance was calculated and represented as ***p < .001, **p < .01, and *p < .05 compared with the control.

Time Considerations

The time needed to establish 3D spheroid growth depends on cell type. For cancer cells, it typically takes 2–3 weeks to establish the number of cells needed for growth and another 2 weeks for 3D spheroid experiments. The antiproliferative activity assay can be done in 3 days, and the assay of 3D tumor growth and inhibition of growth by compounds of interest in 1 week. Once 3D spheroid growth is standardized, the PLA assay can be done in 3–4 days, including imaging under the microscope.

Data Availability Statement

Additional data can be obtained from the author upon request according to the university guidelines on data sharing.