Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields’ locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)–T–based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag–specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients’ glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.

BACKGROUND TTFields has the ability to induce immunogenic cell death (ICD). As immunotherapy and TTFields have different mechanisms of action (MOA), combining these therapies is a rational approach. Contrarily, TTFields may interfere with immune functions critical for effective T cell function. MATERIAL AND METHODS We cultured T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples under normal or TTFields conditions, with or without superantigen-stimulation. In order to assess T cell responses we used eight-color flow cytometry by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Evaluation of direct cytotoxicity was done by using chimeric antigen receptor (CAR) T cells. RESULTS TTFields did not change T cell activation rates for all evaluated functions with the exception of reduced proliferation - in line with TTFields’ MOA. TTFields substantially reduced the viability of activated proliferating T cells, moderately affected activated nonproliferating T cells and had almost no effect on the viability of non-activated cells. Polyfunctionality analysis of T-cells, associated with effective antitumoral responses, demonstrated that under TTFields, the activated non-proliferating T cells retained polyfunctional capabilities. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields. Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes). CONCLUSION The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of antitumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy.

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Background: Combining Tumor Treating electrical Fields (TTFields) with immunotherapy is a rational approach due to their different mechanisms of action (MOA) and to TTFields’ ability to induce immunogenic cell death (ICD). Conversely, TTFields may interfere with immune functions critical for effective T cell responses. Methods: T cells from healthy donors’ peripheral blood or from viably dissociated glioblastoma samples were cultured under normal or TTFields conditions, with or without superantigen-stimulation. Eight-color flow cytometry was used to assess T cell responses by monitoring select pivotal antitumoral functions: proliferation (CFSE), IFNγ secretion, cytotoxic degranulation (CD107a), activation/exhaustion (PD1) and viability. Direct cytotoxicity was evaluated using chimeric antigen receptor (CAR) T cells. Results: The viability of stimulated T cells that attempted to proliferate decreased under TTFields, in line with TTFields’ MOA. Small or no reductions in viability were found in activated T cells that did not attempt to proliferate and in unstimulated T cells. The functionality of stimulated peripheral-blood T cells and tumor-infiltrating T cells (TILs) under TTFields was unhindered: T cells exhibited comparable PD1 upregulation, IFNγ secretion and CD107a expression as controls. T cell polyfunctionality, associated with effective antitumoral responses, was retained under TTFields conditions. PD1-expressing TILs, a subset containing most of the tumor antigen-specific TILs, exhibited unaltered viability and functionality under TTFields. CAR T-cells, which utilize the same killing machinery as unmodified T cells, exhibited unaltered cytotoxic capability under TTFields. Immunohistochemical evaluation of GBM samples before TTFields treatment and after recurrence showed that some patients had accommodated large increases in their CD8 and CD4 counts. RNA-Seq performed on GBM samples from 6 standardly-treated and 6 TTFields-treated patients before treatment and after recurrence. The data shows differential increases in TTFields-treated patients to controls, in the expression of immune genes associated with favorable prognosis (e.g. t-bet, NKG2D, ICOS-L, CD70) and concurrent decreases in genes associated with poor prognosis (e.g. IL4, TSLP, various complement genes). Conclusions: The preclinical data showed that all antitumoral T cell functions examined, but proliferation, were unhindered by TTFields. The clinical data showed that TTFields may shift treated tumors to a state more conducive of anti-tumoral immune responses. Our findings support the further preclinical and clinical investigation into combining TTFields with immunotherapy. Citation Format: Gil Diamant, Hadar Simchony, Tamar Shiloach, Anat Globerson-levin, Lital Gasri Plotnitsky, Zelig Eshhar, Niv Pencovich, Rachel Grossman, Zvi Ram, Ilan Volovitz. Evaluating the compatibility of tumor treating electric fields with key anti-tumoral immune functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3954.