Tumor Treating Fields enhance chemotherapy efficacy by increasing cellular drug uptake and retention in mesothelioma cells

Abstract

Tumor Treating Fields (TTFields) applied with standard chemotherapy have been approved for the first-line treatment of unresectable pleural mesothelioma (PM), an aggressive malignancy with limited effective therapy options. In this study, we demonstrated that the simultaneous exposure to TTFields and doxorubicin or vinorelbine enhanced treatment efficacy in patient-derived PM cells by increasing intracellular drug concentrations. This was achieved by modulating several genes that encode transport proteins, such as the downregulation of P-glycoprotein (P-gp). Using specific, sensitive and quantitative analytical techniques, we observed a more than 70% increase in intracellular concentrations of doxorubicin and vinorelbine in samples treated with TTFields, and a greater than 50% increase in drug uptake in cells exposed to TTFields and pemetrexed. This result indicates that the increased drug concentration observed in TTFields treated cells is significant not only for drugs that are P-gp substrates but also suggests that TTFields could potentially affect other efflux pumps. However, the co-exposure to the drug and TTFields was critical to increasing intracellular drug levels, highlighting the necessity of concurrent use with drugs to enhance the antiproliferative effects of treatment. The in vitro findings were further corroborated by in vivo pharmacokinetic experiments in mice subcutaneously injected with epithelioid PM tumors. Indeed, a 30% increase in intratumor concentrations was observed when vinorelbine was administered with TTFields. Our findings suggest that TTFields could be a well-tolerated approach for enhancing intratumoral drug levels and potentially achieving a more significant therapeutic impact on PM treatment.

Keywords: Pleural mesothelioma; Tumor Treating Fields (TTFields); cancer therapy; cellular drug uptake; combination treatment; pharmacokinetics.

Figure 1

Efficacy of co-treatments. Dose-response curves for DOX and VNR, either alone (black line and symbols) or with TTFields (grey line and symbols), in epithelioid CD473 and sarcomatoid CD60 cells after 72 h exposure. Each symbol represents the mean of four replicates ± standard error. Data were calculated as percentage of controls. The co-treatment was considered as synergistic when its effect value/trendline was below that of the predicted additive effect (dashed light blue line).

Figure 2

Impact of TTFields frequencies on cellular drug uptake. Quantification of intracellular DOX concentration obtained by HPLC analysis after 24 h of treatment with 1 μM DOX, or 24 h of simultaneous exposure to 1 μM DOX and TTFields at different frequencies (100, 150, 200 or 300 kHz). Each column represents the mean of at least four replicates ± standard error (Student’s t test *P<0.05; ***P<0.005; n.s: not significant). None of the observed differences among the cells treated with different TTFields frequencies was statistically significant (Student’s t test P>0.05. One-way ANOVA test P>0.05).

Figure 3

Cellular drug uptake. A. Design of experiment and analysis of intracellular drug concentrations after 24-h exposure to DOX or VNR alone or to two different schedules of treatment with drug and TTFields. Each column represents the mean intracellular drug concentration ± standard error obtained from at least three independent experiments, while symbols represent the single replicates (Student’s t test *P<0.05; **P<0.01. One-way ANOVA test P<0.05 for CD473 cells and P>0.05 for CD60 cells treated with DOX; P>0.05 for CD473 cells and P<0.01 for CD60 cells treated with VNR). B. Representative confocal images of epithelioid CD473 cells labelled with Alexa488-phalloidin (green fluorescence) and untreated or treated with 1 μM DOX (red fluorescence) or DOX and TTFields; scale bar, 20 μm. C. Quantification of intracellular DOX concentration after short treatments with 1 μM DOX or with the simultaneous exposure to DOX and TTFields (2 h of exposure to 150 kHz frequency and 0.76 V/cm intensity for CD473; 2 h and 4 h of exposure to 1.12 V/cm for CD60). Each column represents the mean intracellular drug concentration ± standard error obtained from at least two independent experiments, while symbols represent the single replicates (Student’s t test *P<0.05). D. Quantification of intracellular DOX concentration after different schedules of treatment, DOX (drug alone); TTFields+DOX (sequential exposure); TTFields+I+DOX (sequential exposure with 24-h interval between the two treatments). Each column represents the mean intracellular drug concentration ± standard error obtained from at least three independent experiments, while symbols represent the single replicates (Student’s t test P>0.05).

Figure 4

P-gp modulation and its role in drug efflux. A. Western blot of P-gp expression in epithelioid CD473 cells and sarcomatoid CD60 cells exposed for 24 h and 48 h to TTFields. B. Western blot of P-gp expression after 48 h of TTFields and 24 h after the end of treatment. A and B. The relative density of the band is reported as mean ± standard deviation of at least three independent experiments (Student’s t test *P<0.05; ***P<0.005; ****P<0.001). C. Comparison of drug efflux in epithelioid and sarcomatoid PM cells after 1 h of exposure to 10 μM DOX (black line and symbols) or DOX preceded by 24 h TTFields (dashed line and white symbols). Each symbol represents the mean of at least four replicates ± standard error (Student’s t test *P<0.05; **P<0.01; ***P<0.005). When not visible, error bars are smaller than symbols.

Figure 5

Effects of TTFields on actin expression and polymerization. A. Representative confocal images of epithelioid CD473 and sarcomatoid CD60 cells labelled with Alexa488-phalloidin before (UN, unstimulated) and after the stimulation with 24 h and 48 h of TTFields; white arrows pointed at actin depolymerization; scale bar, 20 μm. B. Western blot of actin levels in CD473 and CD60 cells in basal conditions, after 48 h of TTFields or 24 h after the end of treatment. The relative density of the band is reported as mean ± standard deviation of two independent experiments. Any of the observed differences was statistically significant (Student’s t test P>0.05). C. Quantification of intracellular DOX concentration after 2 h of treatment with 1 μM DOX or with the simultaneous exposure to DOX and 500 nM latrunculin B (LB). The red symbols represent the mean of three replicates (gray and black circles for 1 μM DOX and 1 μM DOX & 500 nM LB, respectively) ± standard deviation (Student’s t test *P<0.05). D. Representative confocal images of sarcomatoid CD60 cells labelled with Alexa488-phalloidin (green fluorescence) after treatment with DOX or DOX and LB. White arrows indicate actin depolymerization; scale bar, 20 μm.

Figure 6

Drug uptake and efflux in PEM treated PM cells. A. Intracellular PEM concentrations after different schedules of treatment. Each column represents the mean of four values ± standard error (Student’s t test *P<0.05. One-way ANOVA test P>0.05 for CD473 cells and P<0.05 for CD60 cells). B. Analysis of drug efflux after 24-h treatment with PEM (black line and symbols) or PEM and TTFields (dashed line and white symbols). Each symbol represents the mean of at least three replicates ± standard error (Student’s t test *P<0.05).

Figure 7

In vivo pharmacokinetics. A. Experimental plan for pharmacokinetic analysis. B. Quantification of VNR concentrations in plasma, liver and tumor. Four mice for each experimental group were sacrificed at 1, 6 and 24 h after drug treatment. Each symbol represents the mean ± standard deviation (Student’s t test VNR vs VNR+TTFields *P<0.05; ***P<0.005).