The central function of the immune system is to protect the integrity of organisms and cells against pathogens and internal threats, such as cancer cells. However, tumors developed numerous strategies to evade immunosurveillance. Cancer immunotherapy has emerged as a promising strategy for treating malignant diseases. The main goal of cancer immunotherapy is to trigger immune responses directed against the tumor. One approach to cancer immunotherapy involves the activation of immunogenic cell death (ICD), which represents a functionally distinct response pattern that triggers the release of damage-associated molecular patterns (DAMPs), tumor antigens, neoantigenes, and other immunostimulatory components from the malignant cells. These diverse signals promote immune cell infiltration into the tumor microenvironment (TME) and enable their activation and maturation.
Inflammasomes are cytosolic multiprotein complexes of the innate immune system that mediate immune responses to pathogens through lytic cell death pyroptosis and release of proinflammatory cytokines. Inspired by their dual action we combined immunogenic cell death with mediators of the immune response, engineering cytokine armed pyroptosis that not only evokes cancer cell destruction but also modulates immune response against the tumor. To induce pyroptosis, we designed tightly regulated gasdermin D (GSDMD) variants with distinct pore-forming capabilities and diverse modes of activation, creating a novel toolbox of ICD effectors that act independently of the endogenous inflammasomes. We demonstrate that GSDMD pore formation in the B16F10 murine melanoma model induces pyroptotic cell death of tumor cells, leading to antitumor immunity and long-lasting remission. The system was further enhanced by the intratumoral expression of immunostimulatory cytokines IL-1β, IL-18 and IL-12, which serve as adjuvants, promoting inflammatory responses and facilitating immune cell recruitment and maturation within the TME. In addition to murine melanoma, our treatment strategy induced tumor regression in the 4T1 mammary carcinoma and CT26 colorectal carcinoma, demonstrating its efficacy across different tumor types, independent of their tissue origin or immunological phenotype. Furthermore, we show that intratumoral induction of cytokine armed pyroptosis elicits systemic antitumor immunity, primarily mediated by natural killer cells and cytotoxic CD8⁺ T cells. Additionally, we employed the Ninjurin 1 (NINJ1) protein to induce ICD in B16F10 tumors, which exhibited antitumor activity comparable to that of GSDMD. Thus, cytokine-armed pyroptosis represents a combinatorial, modulatory, and tumor-agnostic approach for enhancing antitumor immune responses, independent of tumor origin or immune phenotype.
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