Ttfields Induces Immunogenic Cell Death and Sting Pathway Activation Through Cytoplasmic Double-Stranded DNA in Glioblastoma Cells

Abstract

Glioblastoma (GBM) is the most common and deadliest malignant brain cancer in adults despite aggressive chemoradiotherapy. Tumor Treating Fields (TTFields) was recently approved in combination with adjuvant temozolomide chemotherapy for newly diagnosed GBM patients. The addition of TTFields resulted in a significant improvement in overall survival. TTFields are low-intensity alternating electric fields that are thought to disturb mitotic macromolecules’ assembly, leading to disrupted chromosomal segregation, integrity and stability. In many patients, a transient stage of increased peritumoral edema is often observed early during TTFields treatment followed subsequently by objective radiographic responses, suggesting that a major component of therapeutic efficacy by TTFields may be an immune mediated process. We hypothesize that TTFields activates the immune system by triggering pyroptosis and type I Interferon (IFN) response. 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 and quantitative methods including quantitative PCR, ELISA and cytometry. TTFields treatment resulted in a significantly higher rate of micronuclei structures released into the cytoplasm, which were co-localized with two upstream dsDNA sensors AIM2 and interferon-inducible protein cGMP-AMP synthase (cGAS). TTFields-activated micronuclei-dsDNA sensor complexes led to i) induction of pyroptotic cell death, as measured by a specific LDH release assay, and through AIM2-recruited caspase1 and cleavage of pyroptosis-specific Gasdermin D; and ii) activation of STING pathway leading to the release of type I IFNs and pro-inflammatory cytokines downstream of the NFκB pathway. In a co-culture experiment of bone marrow cells or splenocytes with supernatants obtained from GBM cells treated with TTFields, GBM cells depleted of AIM2 or STING or both no longer produced inflammatory cytokines in response to TTFields treatment and supernatants obtained from depleted cells failed to induce bone marrow cells and splenocytes. These results provide compelling evidence that TTFields activates the innate immune system in GBM cells, and a strong rationale for combining TTFields with immune checkpoint inhibitors to create a therapeutic synergy.