The RT4 cell line derives from the explants of a transitional cell papilloma of the bladder (grade 1 carcinoma) and it was obtained from the Rio de Janeiro Cell Bank (BCRJ 0214). Cells were routinely cultured as a monolayer in T-75 flasks (Corning®, New York, USA) with McCoy's 5A Medium with L-glutamine (Sigma-Aldrich®, St. Louis, Missouri, USA) supplemented with 2.2 g/mL of sodium bicarbonate (Sigma-Aldrich®, St. Louis, Missouri, USA), 10% (v/v) Fetal Bovine Serum (Sigma-Aldrich®, St. Louis, Missouri, USA) and 1% (v/v) Penicillin-Streptomycin (10,000 U/mL) (Sigma-Aldrich®, St. Louis, Missouri, USA). The cells were kept in a humidified atmosphere of 5% CO₂ in air at 37°C.

3D spheroids of RT4 cells were generated by two scaffold-free techniques: forced floating method using 96-well round-bottom Ultra Low Attachment (ULA) plates (Corning®, New York, USA) and hanging drop method using 96-well Perfecta3D® Hanging Drop (HD) Plates (3DBiomatrix, Ann Arbor, Michigan). Seeding concentrations of 0.5, 1.25, 2.5, and 3.75 × 10⁴ cells/mL were used to generate 3D spheroids in both methods. For ULA plates it used a working volume of 200 μL of culture medium per well and for HD plates a working volume of 40 μL/well per well. Fresh culture medium (10 μL/well) was added to the HD cultures every 2 days to compensate for losses caused by evaporation. For comparison purposes, RT4 cells were also cultured as a monolayer (2D) in treated 96-well flat-bottom plates (Corning®, New York, USA) at the same seeding concentrations testes in 3D cultures. The morphology of the spheroids, cell growth and metabolism were characterized over a 7-day culture period in quadruplicate.

In order to compare the anticancer effects in 3D spheroids and 2D monolayers, cultures were treated with six different concentrations of doxorubicin: 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 μg/mL in a final volume of 100 μL. Doxorubicin stock solution (1.0 mg/mL) was diluted in McCoy's 5A culture medium to a final concentration of 2X immediately before use. Cultures were also treated with 20% of Dimethyl sulfoxide (DMSO) as a positive control, and fresh culture medium without drug was used as the negative control. Seeding concentrations of 0.5 and 1.25 × 10⁴ cells/mL, and 2.5 and 3.75 × 10⁴ cells/mL were used for ULA plates and HD plates respectively. After 72 h, 3D spheroid cultures were treated with doxorubicin solution by replacing 50% of the supernatant with drug-supplemented culture medium following the protocol established by Friedrich et al. (2009). After treatment, cultures were incubated for 24 h, and analyzed by ATP-based cell viability assays using CellTiter-Glo® 3D (Promega, Madison, Wisconsin, USA). A volume of CellTiter-Glo® 3D solution equal to the volume of the cell culture medium in each well was added. To induce cell lysis, the 96-well plates were vigorously shaken for 5 min, and incubated at room temperature for an additional 25 min. Then, the supernatant was transferred to an opaque-walled multiwell plate, and the luminescence was recorded using a Synergy™ 2 Multi-Mode Microplate Reader (BioTek®, Winooski, Vermont, USA) an integration time of 0.25–1.0 s per well. The same protocol was followed for 24-h monolayer (2D) cultures seeded with the same cell concentration tested in 3D cultures. Six samples per condition (n = 6) were considered for each analysis.

All statistical analyses in this work were performed using Prism software version 7.0 (GraphPad Software, Inc., La Jolla, California, USA). The fold-increase number (FI) and the percentage of apoptotic cells in 2D and 3D cultures were compared using One-way ANOVA statistic method with post-hoc test Tukey HSD and with P < 0.05 considered significant (n = 4). The IC₅₀ values obtained in 2D and 3D cultures were analyzed using Extra-sum-of-squares statistic method with F-test and with P < 0.05 considered significant. To assess the reliability of this statistical analysis, the confidence interval was also determined.

Images obtained by using the phase contrast microscope showed the spontaneous formation of a single cell aggregate per well both in ULA and HD plates in the first 24 h of culture in all cell-seeding concentrations evaluated (Figure 1). After 48 h, a decrease was observed in the size of the cell aggregates, which became more compact, forming solid spheroids. Zanoni et al. (2016) named the time interval for this process as “spheroidization time” that varies depending on the cell type and culture conditions. The RT4 spheroids generated using the ULA and HD plates presented a round-type morphology. According to Kenny et al. (2007), this morphology is related to strong cell-cell adhesion.

SEM images of spheroids from both ULA and HD plates showed that the surface of the spheroids was smooth and continuous (Figures 2A,B). The smooth nature of the surface is attributed to high ECM secretion that conceals individual cells in most parts of the spheroidal surface (Lin et al., 2006). The surface of spheroids from both the ULA and HD plates were covered with plasma membrane projections called microvilli (Figures 2C,D). Surfaces similar to those of tissues with apical microvilli are typical of transitional epithelial cells present in the bladder (Kaufman et al., 2009). Moreover, these microvilli project outward to a high degree in a type of transitional cell carcinoma known as papillary carcinoma, from which the RT4 cell line is derived (Jacobs et al., 1976). Microvilli are not often present in cells cultured conventionally in monolayers (Gardner and Herbst-Kralovetz, 2016; McConkey et al., 2016; Wang et al., 2016), suggesting that RT4 spheroids can reproduce the characteristic features of a papillary tumor in vitro better than monolayer cell cultures.

The roundness of a spheroid is an indicator of the circularity of the projected area of the spheroids. It ranges from 0 to 1.0, and values closer to 1.0 indicate high circularly of the projected area. The roundness of the spheroids from the ULA plates oscillated between 0.75 and 0.95 during the whole experiment (Figure 3A). The best results were obtained for cultures seeded with 0.5 × 10⁴ cells/mL that indicate spheroids with roundness values over 0.90. In HD cultures, spheroids had roundness values over 0.85 and close to 0.90 for most of the time, independent of the cell-seeding concentration (Figure 3D). The low values of roundness observed during the first 24 h of the culture might be due to the lack of total compactness during this period. Throughout the culture, ECM secretions ensure total compactness or “spheroidization,” thus, increasing the roundness of the spheroids. A slight drop in spheroid roundness may occur over time, and is possibly associated with apoptosis and increase in the total spheroidal area (Gong et al., 2015). In general, both methods generated spheroids with a satisfactory circular shape when compared to results reported in literature (Raghavan et al., 2015).

The solidity is an indicator of the regularity of spheroids' surface. According to Santo et al. (2016), tumor spheroids are considered regular if solidity values are higher than 0.90 (no surface edges). The spheroids obtained from the ULA and HD cultures (Figures 3B,E, respectively) can be considered as regular, with solidity values higher than 0.95 after 48–72 h of culture regardless of the cell-seeding concentration used.

According to the sphericity index values, tumor aggregates could be also classified as spherical (SI ≥ 0.90) or non-spherical shape (SI < 0.90), the latter being subdivided into ellipsoidal, “8”-shaped, and irregular (Zanoni et al., 2016). At all cell seeding concentrations tested in the ULA method, the values of the sphericity index (SI) of the spheroids oscillated between 0.80 and 0.90 (Figure 3C). In the case of the HD method, spheroids from cultures seeded with 0.5 and 1.25 × 10⁴ cells/mL had Sphericity Index (SI) values ranging from 0.82 to 0.93 between 48 and 144 h (Figure 3F). During the same period, cultures seeded with 2.5 and 3.75 × 10⁴ cells/mL had SI values ranging from 0.76 to 0.93. In the latter situation, SI values higher than 0.80 were obtained only after 72 h of culture showing that these spheroids take longer to compact compared to spheroids with lower cell-seeding concentrations. Overall, spheroids from ULA and HD plates had values of SI close to 0.90, varying between ellipsoidal and spherical shape. Spheroids with an irregular shape may develop some aberrations such as the presence of two necrotic cores (Zanoni et al., 2016), which is rarely observed in spheroids with a spherical shape. Regular and well-rounded spheroids are more stable when employed in in vitro assays and exhibit less variability (Friedrich et al., 2009; Thoma et al., 2014; Weiswald et al., 2015).

In the ULA method, the spheroid-diameter variation was high in cultures initiated with lower cell concentrations, 0.5 and 1.25 × 10⁴ cells/mL, ranging from 274.08 (±13.98) to 402.01(±34.01) μm and 368.17 (±12.40) to 492.14 (±25.32) μm, respectively (Figure 4A). On the other hand, a low variation in spheroid diameter was observed in cultures initiated with the two highest cell-seeding concentrations. At a seeding concentration of 2.5 × 10⁴ cells/mL, spheroids were obtained, whose diameter was higher than 450 μm at the beginning of the culture and reached values over 500 μm in 120 h. At seeding concentration of 3.75 × 10⁴ cells/mL, the spheroid diameter was larger than 500 μm, during the whole experiment.

3D cell cultures in HD plates were performed using a lower working volume when compared to ULA plates (40 μL for HD and 200 μL for ULA), as recommended by the manufacturer. The cell concentrations tested in HD culture were the same as those used in the ULA culture. However, the total number of cells seeded per well was lower in HD. For this reason, we obtained smaller spheroids by this method when compared to the ULA method (Figure 4B). Spheroids from cultures initiated with 0.5 × 10⁴ cells/mL reached a diameter of only 268.13 ± 11.80 μm by the end of the experiment. Cultures seeded with 1.25 × 10⁴ cells/mL generated spheroids with a diameter higher than 300 μm only after 120 h of culture. Cultures seeded with 2.5 and 3.75 × 10⁴ cells/mL generated spheroids with a diameter ranging from 340.92 ± 16.98 to 430.57 ± 17.12 μm and 330.18 ± 14.62 to 563.97 ± 28.53 μm, respectively. Spheroids with a diameter over 500 μm were not observed. The highest variation in spheroid diameters over 7 days was observed in cultures initiated with 2.5 and 3.75 × 10⁴ cells/mL.

The ideal spheroid diameter for use in cytotoxicity assays is widely discussed in literature, and no agreement has been reached yet. It is known that spheroids with a diameter of up to 200 μm are able to mimic 3D cell-cell and cell-matrix interaction, and they are frequently employed in drug testing (Fehlauer et al., 2005; Lambert et al., 2006; Friedrich et al., 2009). However, small spheroids are inappropriate for creating a pathophysiological condition, since they do not develop chemical or proliferative gradients (Larue et al., 2004; Hardelauf et al., 2011; Patra et al., 2013). Larger spheroids may contain a hypoxic core, and be more heterogeneous, with cells in different proliferation stages (Nath and Devi, 2016). They may also contain a necrotic core, which is be desirable for some drug testing approaches but could be problematic in others (Friedrich et al., 2009). Large tumors in vivo require the development of capillaries that allow blood perfusion in order to maintain cell functions at the center since passive diffusion of nutrients and O₂ is insufficient (Alemany-Ribes and Semino, 2014). Without a perfusion system in vitro, spheroids with a diameter >500 μm develop a secondary necrosis core, which makes pathophysiological conditions complicated and difficult to control, subsequently, affecting the accuracy of the cytotoxicity assay (Inamdar and Borenstein, 2011; Wan et al., 2016). For these reasons, several researchers choose to work with spheroids of diameter ranging between 300 and 500 μm (Friedrich et al., 2009; Hardelauf et al., 2011; Wen et al., 2013; Ivanov et al., 2014; Patra et al., 2016; Wan et al., 2016). In the ULA method, 300–500 μm RT4 spheroids were obtained from cultures initiated with 0.5 and 1.25 × 10⁴ cells/mL. On the other hand, in the HD method, the cultures initiated with 2.5 and 3.75 × 10⁴ cells/mL generated spheroids with the appropriate diameter (300–500 μm).

Cell growth was observed at all cell-seeding concentrations for both the ULA and HD methods. In ULA cultures a fold increase (FI) of 7.0 ± 1.11, 4.5 ± 0.87, 3.5 ± 1.0, and 2.7 ± 0.33 in cell concentration was observed for cultures initiated with 0.5, 1.25, 2.5 and 3.75 × 10⁴ cells/mL, respectively (Figure 5A). Viability of over 90% was maintained during most of the culture period in cultures seeded with 0.5 and 1.25 × 10⁴ cells/mL (Figure 5B). The viability of spheroids from the two highest cell-seeding concentrations, 2.5 and 3.75 × 10⁴ cells/mL was maintained near 90% only during the first 96 h, and decreased to values lower than 70% by the end of the culture period.

Spheroids cultured in HD plates showed a FI of 7.0 ± 3.34, 5.5 ± 1.82, 5.65 ± 0.91, and 4.64 ± 0.52 in cell concentration in cultures seeded with 0.5, 1.25, 2.5, and 3.75 × 10⁴ cells/mL, respectively (Figure 5C). No significant differences were observed between FI numbers of ULA and HD cultures. Viability of over 90% was maintained during the whole experiment in cultures seeded with 2.5 and 3.75 × 10⁴ cells/mL (Figure 5D). Cultures seeded with 0.5 and 1.25 × 10⁴ cells/mL showed a small drop in viability after 72 h of culture, but all cultures showed viability of over 80% by the end.

When cultured in monolayers (2D), RT4 cells showed a typical in vitro cell-growth profile, having the lag, exponential, and stationary phases in cultures seeded with 2.5 and 3.75 × 10⁴ cells/mL (Figure 5E). This cell-growth profile was not observed in 3D cultures. 2D cultures showed a FI of 22.1 ± 2.61, 19.5 ± 2.58, 16.7 ± 1.0, and 12.4 ± 2.2 in cell concentration when seeded with 0.5, 1.25, 2.5, and 3.75 × 10⁴ cells/mL, respectively (Figure 5F). These values were higher than those observed for 3D cultures (One-way ANOVA with post-hoc test Tukey HSD, P < 0.001). Under all conditions, viability was maintained close to or higher than 90% during the first 72 h, and showed a small drop after this point, but values over 80% were maintained by the end (Figure 5B).

The differences in growth rate observed between 3D and 2D cultures with RT4 cells are also observed in other cell lines (Maria et al., 2011; Chitcholtan et al., 2012; Luca et al., 2013). All cells in 2D cultures receive equal amount of nutrients and growth factors from the medium, since they attach themselves to a flat surface, forming a monolayer (Huang et al., 2013). During the exchange of culture medium, most of necrotic cells are removed, and therefore, proliferating cells are predominant in 2D cultures (Edmondson et al., 2014). On the other hand, 3D spheroids consist of cells in different stages with viable and proliferating cells only in the outer layers. This feature results in cell growth dynamics with initial cell proliferation followed by delayed growth, which is commonly observed in in vivo tumors (Sutherland, 1988). Therefore, when cultured as spheroids, RT4 cells were able to exhibit the growth characteristics observed in bladder tumor cells in vivo.

Apoptotic cells in 2D and in 3D cultures were also evaluated by flow cytometry using Annexin V-FITC as the apoptosis marker. The percentage of apoptotic cells at 120 and 168 h of culture in each situation is shown in Figure 6. For 120-h cultures, statistical difference was only observed between 2D and 3D ULA cultures seeded with 2.5 × 10⁴ cells/mL (One-way ANOVA with post-hoc test Tukey HSD, P < 0.05). In case of 168-h cultures, we observed a significant higher number of apoptotic cells in HD cultures seeded with 0.5, 2.5, and 3.75 × 10⁴ cells/mL when compared with 2D cultures (One-way ANOVA with post-hoc test Tukey HSD, P < 0.05). Apoptosis may occurs over time in these systems due to the lack of a vasculature (Hirschhaeuser et al., 2010; Gong et al., 2015; Stock et al., 2016). A high number of apoptotic cells could be also an indicator of dysfunctional mechanisms of cell-cell and cell-ECM interactions, since these interactions operate to control apoptosis (Zahir and Weaver, 2004).

The metabolic activity of RT4 cells in 3D cultures was also characterized in this study, and compared with the results obtained under 2D culture conditions. Under 2D, the consumption of glucose by RT4 cells was only observed in cultures with the two highest cell-seeding concentrations (Figure 7A). In cultures seeded with 2.5 and 3.75 × 10⁴ cells/mL, the concentration of glucose decrease from 17 ± 0.16 mM to 7.5 ± 0.32 and 4.35 ± 1.12 mM, respectively. As lactate is a byproduct of glucose metabolism, the concentration of lactate reached the highest level, about 30 mM, in the cultures with the higher glucose consumption (2.5 and 37.5 × 10⁴ cells/mL; Figure 7B). On the other hand, a decrease in glutamine concentrations in the medium was observed, regardless of the cell seeding concentration (Figure 7C). Glutamine levels decreased from 1.54 ± 0.01 mM to 0.68 ± 0.02, 0.40 ± 0.02, 0.14 ± 0.06, and 0.10 ± 0.01 mM in cultures initiated with 5, 1.25, 2.5, and 3.75 × 10⁴ cells/mL, respectively. Lea and collaborators have already described that RT4 cells exhibited glutamine-dependent cell growth, unlike other bladder cancer cell lines, wherein glucose is the main energy source (Lea et al., 2015).

The consumption of nutrients of RT4 cells in 3D cultures was lower when compared to 2D culture. This situation is expected once metabolism can be altered depending on the type of cancer cells, invasiveness and culture conditions (Conde et al., 2015). Glucose was not consumed neither in ULA cultures nor HD cultures (Figures 7D,G) resulting in a small production of lactate in the culture medium (Figures 7E,H). The variations in glucose levels in the 3D cultures, mainly in HD method, can be explained by the considerable medium evaporation (around 50% after 7 days of culture). Although the consumption of glutamine in 3D cultures was lower than 2D cultures, a preference for glutamine instead of glucose was also observed (Figures 7F,I). The reduction in metabolic activity of cells within multicellular spheroids was also observed in other studies (Desoize and Jardillier, 2000). This must be related to the fact that metabolic enzymes involved in glycolysis and glucose transporters are downregulated when cells are organized into 3D spheroids (Werner and Werb, 2002). Another interesting fact is that the 3D architecture may induce a sustained effect on cellular metabolism, since some works have shown that single cells derived from dissociated spheroids exhibited consistently reduced metabolism (Zahir and Weaver, 2004). The decreased dependence on glycolysis or enhanced oxidative phosphorylation would provide cells less sensitive to eventual changes in nutrition when cultured in 3D spheroids (Zahir and Weaver, 2004).

The drug response study was conducted with spheroids from 72 h-cultures with cell-seeding concentrations of 0.5 and 1.25 × 10⁴ cells/mL for ULA plates, and 2.5 and 3.75 × 10⁴ cells/mL for HD plates. Under these conditions, spheroids with the desired diameter (300–500 μm) and shape parameters were obtained. Spheroids and monolayer cultures were treated with different concentrations of doxorubicin: 4.0, 2.0, 1.0, 0.5, 0.25, and 0.125 μg/mL. Doxorubicin is a chemotherapeutic agent used in the treatment of several types of tumors (Carter and Comis, 1975), including bladder cancer. Therefore, doxorubicin was chosen to evaluate the sensitivity of RT4 cells in the 3D cultures. Images obtained by phase contrast microscopy, after the 24-h treatment with doxorubicin showed a cloud of debris and dying cells surrounding the spheroids at all drug concentrations, while most of cells in 2D cultures were dead, as represented in Figure 8.

Differences in drug response between cells of 2D and 3D cultures could be observed in all situations as shown in Figure 9. The inhibitory dose-response curve fit showed that the activity of doxorubicin was higher in 2D cultures than in 3D cultures. These results were confirmed by the IC₅₀ values (Table 1). In both 3D conditions, ULA and HD, the IC₅₀ values were statistically higher than those observed in 2D cultures (P < 0.001 for cultures seeded with 0.5, 1.25, and 3.75 × 10⁴ cells/mL, P < 0.05 for cultures seeded with 2.5 × 10⁴ cells/mL, extra-sum-of-squares F-test).

Some studies have compared the effect of anticancer drugs in 2D and 3D cultures using different cancer cell lines (Adcock et al., 2015; Baek et al., 2016; Oliveira et al., 2016; Wan et al., 2016). Most of them have already pointed that tumor cells in 2D cultures often die after treatment with the anticancer drug, while cells in 3D cultures are often more resistant (Ponce de León and Barrera-Rodríguez, 2005; Hongisto et al., 2013; Breslin and O'Driscoll, 2016). These differences can be attributed to several reasons. The first reason is concerned with the difference in the accessibility of the drug to cells (Edmondson et al., 2014) once exposure to medium/drugs is different between 2D and 3D cultures. Cells in monolayer are uniformly exposed to drugs present in the medium, whereas drug may not be able to fully penetrate the 3D structure (Kim, 2005; Yip and Cho, 2013). Besides diffusion, the pH of the cells is also important since low pH reduces drug uptake, thus, contributing to drug resistance (Swietach et al., 2012). It has been shown that the hypoxic microenvironment at the core of the spheroids leads to the activation of genes involved in cell survival and drug resistance (Trédan et al., 2007). Since doxorubicin promotes cellular death through the formation of reactive oxygen species (Wartenberg et al., 2005), this drug would have a lower therapeutic efficacy under hypoxic conditions. The differences in cell phases are also important in drug resistance. In monolayer cultures, there is a predominance of proliferating cells, whereas in 3D cultures, there is a combination of cells at different stages (heterogeneity). This fact is important since some drugs such as doxorubicin are effective only against actively proliferating cells (Chitcholtan et al., 2012; Wen et al., 2013). Finally, the fifth reason is the difference in expression levels of oncogenes that may be involved in drug action and affect its efficacy (Edmondson et al., 2014).

A comparative analysis of the main results obtained employing ULA and HD plates for 3D culture of RT4 cells is presented in Table 2. Both methods, ULA and HD, generated RT4 spheroids between 24 and 48 h of culture (spheroidization time) with a round-type morphology and the presence of compact surface containing microvilli. Regarding the shape, we generated ellipsoidal/spherical spheroids (sphericity index around 0.90) with a regular surface (solidity higher than 0.95) in both methods. On the other hand, spheroids from ULA plates showed roundness values more close to 1.0 than spheroids from HD plates.

The growth of cells in the spheroids was assessed according to changes in diameter, cell number, and metabolism. This information was important in selecting the cell-seeding concentration that was used for the drug resistance assay. A reduced cell growth was observed in spheroids compared to 2D cultures. HD plates showed a significant higher number of apoptotic cells when compared to 2D in 168-h cultures. Neither nutrient exhaustion nor accumulation of lactate to inhibitory levels was observed in the culture medium. The IC50 values obtained in both 3D cultures were similar and significantly higher than those obtained to 2D cultures.

The necessity to transfer the spheroids from the HD plates to the conventional 96-well plates to carry out the cytotoxicity assay was considered a limitation of this method. Maintaining the culture conditions of the HD method was also considered a limitation since the low working volume resulted in the quick evaporation of culture medium, and any mechanical shock during manipulation and culture would shatter the spheroids. The ULA method, on the other hand, offers a simpler workflow, and can be easily used in high-through put screening platforms. Based on these limitations, we considered the forced floating method using ULA plates as the most suitable and straightforward method to generate RT4 spheroids for cytotoxicity assays. Our findings corroborate with the literature reporting promising results using ULA method with different types of tumor cells (Vinci et al., 2012; Howes et al., 2014; Feng et al., 2017; Selby et al., 2017).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.