Deconstructing synthetic inflammasomes in tumors drives diverse antitumor responses
Inflammasomes are cardinal defense machines that utilize cytokine and immunogenic cell death components to engage the immune system against pathogens. On the other hand, inflammasomes have ambiguous and context-dependent roles in cancers that are known to downregulate effectors of immunogenic cell death. In this study, we deconstructed inflammasomes by introducing one or both inflammasome signaling arms in diverse types of tumors regardless of the expression of endogenous inflammasome components. To induce immunogenic cell death, we designed tightly regulated GSDMD variants comprising different pore-forming capabilities and diverse modes of activation.  We show that electroporation of plasmids encoding the pyroptotic component into B16 melanoma tumors leads to tumor regression and complete remission in a quarter of treated animals. The inflammatory cytokines IL-1ß and IL-18, along with the T-cell activator IL-12, did not provide protection when produced solely within tumors. However, they significantly boosted anti-tumor immunity when combined with pyroptosis. Careful selection of immunostimulatory molecules is imperative as a combination of IL-1ß and IL-18 or the introduction of IFN? antagonizes the protective action of pyroptosis by upregulation of several immune checkpoints. Additionally, we show that deconstruction of inflammasomes locally, without inducing systemic inflammation, provides protection against distant tumors and proves effective across various tumor types. Deconstructed inflammasomes are thus a powerful, tunable and tumor-agnostic strategy to enhance antitumor responses to even the most resilient types of tumors. Overall design: 2×10e6 B16F10 Red-FLuc cells were subcutaneously inoculated into the right flank of C57BL/6J OlaHSd mice. When tumors were palpable and reached approximately 50 mm3, determined using the volume formula (width x length x height x p)/6, 20 µg of pcDNA empty vector, mIL-1ß, mIL-18, single chain mIL-12 or IFN? plasmid were injected into the tumor followed by electroporation using Gene Pulser Xcell Total Electroporation System (Bio-Rad Laboratories) with the parameters of Voltage 900, pulse width 100 µs, and pulse number 5. When the combination of mIL-1ß and mIL-18 was used, 10 µg of each plasmid was electroporated. Two and six days after the first electroporation, tumors were injected with 20 µg of pcDNA empty vector or NT GSDMD plasmid and electroporated using the same parameters. We collected tumor samples 14 days post B16 inoculation and total RNA was extracted from the fresh tumors using TriPure Isolation Reagent (Roche) according to the manufacturer's instructions. Collected RNA from the animals of the same group was pulled together and sent in triplicates for bulk RNA sequencing.