Physiological Cell Culture Media Tune Mitochondrial Bioenergetics and Drug Sensitivity in Cancer Cell Models

Abstract

Two-dimensional cell cultures are established models in research for studying and perturbing cell-type specific functions. However, many limitations apply to the cell growth in a monolayer using standard cell culture media. Although they have been used for decades, their formulations do not mimic the composition of the human cell environment. In this study, we analyzed the impact of a newly formulated human plasma-like media (HPLM) on cell proliferation, mitochondrial bioenergetics, and alterations of drug efficacies using three distinct cancer cell lines. Using high-resolution respirometry, we observed that cells grown in HPLM displayed significantly altered mitochondrial bioenergetic profiles, particularly related to mitochondrial density and mild uncoupling of respiration. Furthermore, in contrast to standard media, the growth of cells in HPLM unveiled mitochondrial dysfunction upon exposure to the FDA-approved kinase inhibitor sunitinib. This seemingly context-dependent side effect of this drug highlights that the selection of the cell culture medium influences the assessment of cancer drug sensitivities. Thus, we suggest to prioritize media with a more physiological composition for analyzing bioenergetic profiles and to take it into account for assigning drug efficacies in the cell culture model of choice.

Keywords: cancer cells; cell bioenergetics; cell culture media; cell proliferation; kinase inhibitor; mitochondrial function.

Figure 1

Media formulation of RPMI, DMEM, and HPLM. (a) All experiments were performed in these media supplemented by 10% dialyzed FBS. Glucose concentration of DMEM and RPMI was modified as physiological concentration (low glucose, 5 mM). (b) The chart shows the relative concentrations of RPMI and DMEM media supplemented with 5 mM glucose compared with HPLM. Components not present in RPMI and DMEM but present in HPLM are denoted as “absent”; components not present in the HPLM but present in the other media are denoted as “present”.

Figure 2

HPLM and RPMI/DMEM effects on cell growth of SW620, MCF7, and A375 cells. (a–c) Proliferation of SW620 cells grown in RPMI^+FBS or HPLM^+FBS. Growth curves are shown in (a), AUC are plotted in (b), and slope variations are shown in (c). (d–f) Same analyses performed for MCF7 cells grown in either RPMI or HPLM showing proliferation curves (g), AUC (h), and slope variation (i). (g–i) Proliferation of the A375 cell line grown either in DMEM^+FBS or HPLM^+FBS. In proliferation curves and AUC, values are represented as median ± IQR (50% range). Unpaired non-parametric t-test analysis; N = 5.

Figure 3

HPLM alters mitochondrial function in SW620, MCF7, and A375 cell populations when compared to standard cell culture media. (a,c,e) Representative respiratory traces for coupling control protocol SUIT-003-D009 in (a) SW620, (c) MCF7, and (e) A375 cells. Blue lines: O₂ concentration [µM]; dark orange lines: O₂ flow per cell [amol × s⁻¹ × x⁻¹] in cells grown and measured in (a,c) RPMI^+FBS or (e) DMEM^+FBS; light orange lines: O₂ flow per cell [amol × s⁻¹ × x⁻¹] in cells grown and measured in HPLM^+FBS. “x” represents the unit cell. Sequential steps: cell addition, oligomycin (Omy), uncoupler CCCP (U), rotenone (Rot) and antimycin A (Ama). Total ROUTINE respiration (R’_tot), LEAK respiration (L’_tot), and ET capacity (E’_tot) not baseline-corrected for residual O₂ consumption (Rox). (b,d,f) O₂ flow (R, L, E) amol × s⁻¹ × x⁻¹ ] baseline-corrected for Rox in (b) SW620, (d) MCF7 and (f) A375 cell lines. (g–i) Flux control ratios FCR in SW620, MCF7, and A375 cells lines. Results are represented as median ± IQR (50% range). 2-way ANOVA with Bonferroni’s multiple comparison test or non-parametric unpaired t-test analysis; N = 4–5.

Figure 4

Bioenergetic cluster analyses in different respiratory coupling states of the SW620, MCF7, and A375 cell lines. BCA plots of steady state conditions (a–h). Plots of L over R, R over E, and L over E. (a–c) MCF7 showing two heterolinear bioenergetic clusters in RPMI^+FBS (c1) and HPLM^+FBS (c2). This indicates changes in mitochondrial quality between both media. (e–g) A375 showing two isolinear bioenergetic clusters in DMEM^+FBS (c1) and HPLM^+FBS (c2). Proportionality with zero intercept indicates changes of mitochondrial density in the cells grown in the two media. (d,h) Coupling efficiencies (E-L)/E over ET capacities in, MCF7 (d) and A375 (h). The lower (E-L)/E values in MCF7 cells in RPMI^+FBS at lower ET values indicate a drop in mitochondrial quality with RPMI^+FBS. In A375, the (E-L)/E values remain constant over ET in both media indicating that mitochondrial quality is preserved.

Figure 5

Differential effects of sunitinib on cell respiration of SW620 cells grown in RPMI or HPLM. (a,b) Representative respiratory traces for coupling control protocol SUIT-003-D009 in SW620 cells grown in (a) RPMI^+FBS and (b) HPLM^+FBS, and exposed to 10 µM sunitinib for 3 h. Blue lines: O₂ concentration [µM]; light orange lines: O₂ flow per cell amol × s⁻¹ × x⁻¹ ] in DMSO control; and dark orange lines: O₂ flow per cell [amol × s⁻¹ × x⁻¹] in sunitinib-treated cells. “x” represents the unit cell. (c,d) O₂ flow (R, L, E) [amol × s⁻¹ × x⁻¹] baseline-corrected for Rox in (c) RPMI^+FBS and (d) HPLM^+FBS. (e,f) Flux control ratios FCR in SW620 cells grown in (e) RPMI^+FBS and (f) HPLM^+FBS and exposed to sunitinib (chemical structure depicted). Median ± IQR (50% range). 2-way ANOVA with Bonferroni´s multiple comparison test; N = 5–6.

Figure 6

Bioenergetic cluster analyses in different respiratory coupling states of the sunitinib-exposed SW620 cells. Plots of L over R, R over E, and L over E. (a–c) SW620 cells grown in HPLM^+FBS and exposed to either DMSO or 10 µM sunitinib for 3 h. In this case, two heterolinear clusters (c1, DMSO and c2, sunitinib) were detected when L is plotted over E, one with higher L when E is decreasing (c2) indicating dyscoupling induced by sunitinib. (c) Coupling efficiencies (E-L)/E over ET capacities in SW620 cells grown HPLM^+FBS (d), and exposed to either DMSO or 10 µM sunitinib for 3 h. The two clusters, one with lower (E-L)/E values in cells grown in HPLM^+FBS and exposed to sunitinib (c2) indicates a drop in mitochondrial quality with the sunitinib treatment. Sunitinib chemical structure is depicted for drug-exposed SW620 cells (a–d).