Tumor-treating fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy

Abstract

Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100-300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.

Keywords: Anti-PD-1; Autophagy; ER stress; Immunogenic cell death; Tumor-treating fields.

Conflict of interest statement

We wish to disclose that TV, NK, SD, YP, AS, MM, RSS, CTB, EZ, RB, SC, AI, MG, EDK, AK, and UW are employees of Novocure and hold stock in Novocure Ltd. Yoram Palti holds stocks in Novocure Ltd. Novocure holds several patents related to TTFields. The authors declare that there are no other conflict of interest.

Figures

Fig. 1

TTFields kill cancer cells by triggering apoptosis followed by extracellular release of HMGB1. LLC-1 and murine CT-26 colon carcinoma cells were treated with TTFields for 24–72 h at optimal frequency. a Effect of TTFields treatment on cell counts. N ≥ 3, and data are presented as mean ± SD. b Elevation of percentage of early apoptotic cells (Annexin V +/7AAD-) and late apoptotic cells (Annexin V+/7AAD+) following treatment with TTFields. N ≥ 3. c Release of HMGB1 was monitored using ELISA assay. N ≥ 3, and data are presented as mean ± SD. P values were determined using one-way ANOVA followed by Dunnett’s post-test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

TTFields application mediate cell surface exposure of calreticulin. LLC-1 and CT-26 cells were treated with TTFields for 24–72 h at optimal frequency. A quantitative analysis of CRT surface exposure was performed using flow cytometry. a Representative plots of flow cytometry analysis of CRT, b CRT surface exposure among viable cells (7AAD-). c Median fluorescence intensity of CRT in viable cells. In b and cN ≥ 3, and data are presented as mean ± SD, d Upper panel—Immunoblot analysis of peIF2α. Lower panel—densitometric analysis (arbitrary units normalized on the expression of the housekeeping protein Vinculin). N ≥ 3, and data are presented as mean ± SEM; P values were determined using one-way ANOVA followed by Dunnett’s post-test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

TTFields induce autophagy-dependent reduction in intracellular ATP levels. LLC-1 cells were treated with TTFields for 24–72 h at optimal frequency. a Flow cytometry measurements of quinacrine staining in viable cells (7AAD-) as indicator of intracellular ATP levels (Upper panel). N ≥ 3, and data are presented as mean ± SD. Alternatively, ATP release induced by TTFields was measured by enzymatic methods (lower panel). N = 2, the results are from one representative experiment, and data are presented as mean ± SD. b Ultrastructural scanning transmission electron microscope analysis of LLC-1 cells treated with TTFields for 48 h. Autophagosomes (Red arrows) and autolysosomes (Blue arrows) are indicated. Magnification ×6000, ×15000. c Immunofluorescence staining for LC3 (green) and DAPI (blue; left panel). Magnification ×40. Quantification of LC3II signal per cell (right panel). N = 4, and the results are reported as mean ± SD of pooled independent experiments. d LLC-1 cells were either left untreated, or treated with TTFields for 24–72 h. CQ (20 µM) was added 4 h before cells were collected. Samples were immunoblotted for LC3 and GAPDH. N = 2, and the results are reported as mean ± SD of pooled independent experiments. P values were determined using one-way ANOVA followed by Dunnett’s post-test for (a-upper panel, d) or unpaired two-tailed t test for (a-lower panel, c). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

Culture of TTFields-treated cancer cell suspension induces maturation of bone marrow-derived dendritic cells. a, b Co-culture of cell tracker deep red labeled, untreated, or TTFields-treated LLC-1 cells and BMDCs. CD11c+Deep Red+events were quantified using flow cytometry. a Representative flow cytometry plot and b quantification of phagocytosis index. N = 3, and data are presented as mean ± SD. c–e Co-culture of untreated, TTFields-treated, F/T-treated, or LPS-treated LLC-1 cells and BMDCs. BMDCs maturation phenotype was assessed using flow cytometry. Quantification of activation markers: c MHC II (D-dim, I-intermediate, and B-bright expression), d CD80, and e CD40. N ≥ 4, and data are presented as mean ± SEM. f LLC-1 cells were either left untreated or treated with TTFields- (48 h), or F/T-treated cells and were intraperitoneally injected into wild-type C57/Bl6 mice. Equal volumes of PBS were injected in mice as negative controls. Peritoneal exudate cells were collected 48 h later and analyzed using flow cytometry. Graphs represent the number of CD45 + cells in peritoneal exudate cells. N = 2, bars indicate mean of two pooled independent experiments with 5–7 mice per group. Each circle represents one mouse. P values were determined using unpaired two-tailed t test for (b) or one-way ANOVA followed by Dunnett’s post-test (c–f). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

TTFields in combination with anti-PD-1 are therapeutically effective in murine lung cancer model. Ten-12-week-old male C57Bl/6 mice were injected directly into the lungs with Lewis lung carcinoma (LLC-1; 3 × 103 cells). a Application of TTFields to the mouse lungs was initiated 6 days afterward and was maintained for 7 days. Mice received an I.P. injection of anti-PD-1 (αPD-1) or Rat IgG2a, as indicated in the scheme. b At the end of the experiment, tumor volume was measured using Vernier calipers. N = 3, and the results are reported as mean ± SD of three pooled independent experiments with total of 5–8 mice per group. Tumors were harvested and analyzed for the percentages (c–e) and PD-L1 expression levels (f–h) of tumor (c, f) CD45+ cells, d, g dendritic cells (CD45+CD11c+), e, h macrophages (CD45+CD11b+F4/80+) using flow cytometry. Percentages of i CD3+CD8+, j CD3+CD4+, and k Foxp3+CD3+CD4+TILs. l, m TILs were harvested from LLC-1 tumor-bearing mice and stimulated with anti-CD3 and anti-CD28 before intracytoplasmic cytokine staining. Expression levels of IFN-γ+in CD3+CD8+ (l) and CD3+CD4+ (m). N = 2, and bars indicate mean of two pooled independent experiment with 5–8 mice per group. Each circle represents one mouse. P values were determined using the Kruskal–Wallis test followed by a Dunn’s post-test for (b) or unpaired two-tailed t test for (c–m). *P < 0.05; **P < 0.01; ***P < 0.001. MFI Median fluorescence intensity

Fig. 6

TTFields in combination with anti-PD-1 are therapeutically effective in murine colon cancer model. a Ten-week-old female Balb/c mice bearing 60 mm3 subcutaneous CT-26 tumors were treated with TTFields for 14 days, with a 3-day break (days 13–16). Mice received an I.P. injection of anti-PD-1 (αPD-1) or Rat IgG2a, as indicated in the scheme. b At the end of the experiment, tumor volume was measured using Vernier calipers. N = 2, and the results are reported as mean ± SD of two pooled independent experiments with total of 5–11 mice per group. Tumors were harvested and analyzed using flow cytometry for the percentages (c–e) and PD-L1 expression levels (f–h) of tumor (c, f) CD45+ cells, d, g dendritic cells, e, h macrophages. Percentages of i CD3+CD8+, j CD3+CD4+, and k Foxp3+CD3+CD4+TILs. P values were determined using two-way ANOVA with Tukey’s post-test for (b) or unpaired two-tailed t test for (c–k). *P < 0.05; **P < 0.01; ***P < 0.001