焦亡来源的细胞外囊泡作为近红外光免疫治疗中的关键免疫激活因子

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.