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OBJECTIVES Tumor Treating Fields (TTFields) was approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed Glioblastoma (GBM) patients and resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly. In many patients, a transient stage of increased peritumoral edema is often observed early during TTFields treatment, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. We hypothesize that TTFields activate the immune system by triggering pyroptosis and type I Interferon (IFN) response. METHODS A panel of GBM cell lines were treated with TTFields at the clinically approved frequency of 200 kHz using an in vitro TTFields system. Cells were analyzed for the production of micronuclei and activation of both pyroptosis and STING pathways using immunostaining, quantitative PCR, ELISA and cytometry. Pre-treated mouse GBM cells were injected into mouse brain to monitor survive and immunophenotyping. GBM patients’ blood was collected, and PBMC were isolated and analyzed by single cell RNAseq. RESULTS TTFields resulted in a significantly higher rate of micronuclei structures released into the cytoplasm, which were co-localized with two upstream dsDNA sensors AIM2 and cGAS. TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by LDH release assay, and through AIM2-recruited caspase1 activation and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway leading to the increase of type I IFNs and pro-inflammatory cytokines. In mouse model, double knocking down of STING/AIM2 eliminated the tumor suppression effects caused by TTFields. TTFields pretreated wild type cells successfully elevated dendritic cell level in mouse cervical lymph nodes which can be reversed by double knocking down. CONCLUSIONS These results provide compelling evidence that TTFields induces effective anti-tumor immunity in GBM cells and patients. |
