Pyroptosis-derived extracellular vesicles as key immunoactivators in near-infrared photoimmunotherapy

Background: Pyroptosis, a lytic and highly inflammatory form of programmed cell death, has emerged as a powerful mechanism for augmenting anti-tumor immunity. Although antibody drug conjugate (ADC)-mediated near-infrared photoimmunotherapy (NIR-PIT) can induce tumor-specific pyroptosis and enhance immune responses, the downstream mediators that bridge pyroptotic cell death to immune activation remain undefined. Here, we investigated whether pyroptosis-derived extracellular vesicles (Pyro-EVs) constitute a key immunostimulatory output of tumor pyroptosis and sought to define their molecular composition and immune-activating functions. Methods: Pyroptosis was induced in SCC7 murine carcinoma cells using Cetuximab-IR700, a clinically-approved NIR-PIT ADC. EVs released during pyroptosis were isolated by ultracentrifugation and characterized by NTA and nano-flow cytometry. Deep proteomic profiling with organelle-origin mapping, GO enrichment, and nuclear-cytoplasmic signature scoring was performed to define Pyro-EV identity. DNA cargo was examined by nano-flow cytometry, DNase-protection assays, and confocal microscopy. Immunological activity was evaluated by exposing macrophages to Pyro-EVs, followed by RNA-seq, ELISA, and Western blot assessment of nucleic-acid sensing pathways. Results: NIR-triggered pyroptosis induced rapid release of a distinct EV population, generating Pyro-EVs at 2-3x the abundance of basal EVs. Proteomics identified a pyroptosis-specific signature with 726 emergent proteins enriched for ribosomal subunits, chromatin components, and DNA-binding regulators, absent in apoptotic or necrotic EVs. These molecular hallmarks indicate nuclear rupture and cytoplasmic mixing, establishing Pyro-EVs as a unique class of lytic cell-death-derived vesicles. Pyro-EVs carried elevated levels of double-stranded nuclear DNA, confirmed by DNase resistance and colocalization with chromatin-binding proteins. Functionally, Pyro-EVs were strong activators of innate immunity, inducing broad M1-polarizing programs in macrophages. RNA-seq showed induction of TLR7/9, MyD88, IRAK1/4, and NF-κB-related genes; Western blot validated activation of the TLR7/9-MyD88-IRAK-p65 axis, and ELISA demonstrated increased secretion of TNF-alpha, IL-6, and other cytokines, confirming DNA-sensing-dependent innate immune activation. Conclusions: This study identifies Pyro-EVs as a previously unrecognized, immunogenic vesicle class that emerges during NIR-PIT. Pyro-EVs are rich in nuclear DNA and chromatin-associated factors and serve as high-potency activators of macrophage TLR7/9 signaling, establishing them as a central mediator connecting pyroptotic tumor death to innate immune amplification. These findings redefine the immunobiology of pyroptosis and uncover Pyro-EVs as a mechanistic driver of pyroptosis-induced anti-tumor immunity.