Tumor-derived extracellular vesicles as key messengers of radiotherapy-induced immunogenic cell death to sensitize small cell lung cancer to immunotherapy

Background: We previously showed low-dose radiotherapy (LDRT) exerts immunostimulatory effects in both preclinical models and clinical settings for SCLC, yet its immunogenic cell death (ICD) mechanisms remain unclear. As extracellular vesicles (EVs) mediate intercellular stress signaling, we hypothesized that tumor-derived EVs play a critical role in orchestrating immunogenic signaling in the initial stages of radiotherapy response. This study tracks ICD signals from tumor to DCs via EV monitoring and elucidate their function spectrum throughout this process. Methods : Plasma EVs from prospective extensive-stage-SCLC cohorts treated with LDRT plus chemo-immunotherapy and PDX/PDO-derived EVs post-irradiation underwent proteomic profiling. Post-irradiation cellular components - cell pellets, debris, and EVs - were used to activate DC-T axis respectively. EV inhibition and live-cell imaging confirmed radiation-induced EVs (RT-EVs) activate the axis versus non-irradiated EVs (NT-EVs). SCLC CDX and PDX models were established, and corresponding homogeneous PBMCs were collected for in vivo EV function evaluation.An ICD tracking system based on stable isotope labeling traced EV-mediated immunogenic transfer from irradiated SCLC and MC-38ova cells to DCs. Results : Proteomic analysis identified LDRT-EVs were specifically enriched in DC activation pathways and exhibited stronger ICD hallmarks, termed “Spark-EVs”. Spark-EVs significantly enhanced DC and T cell activation more than other cellular components, with efficacy comparable to whole-cell lysates. Spark-EVs potently activated the DC-T cell axis compared to NT-EVs, whereas the EV release inhibitor reversed this effect to control levels. Live-cell imaging confirmed nearly threefold greater DC uptake of Spark-EVs than NT-EVs (p < 0.0001). In CDX models, Spark-EV-pulsed DCs + anti-PD-1 enhanced tumor suppression versus NT-EVs + anti-PD-1 or anti-PD-1 alone. Patient T cells primed with Spark-EV-pulsed hDCs showed enhanced stemness and cytotoxicity in vitro. Adoptive transfer of these T cells into PDX models yielded superior tumor control. EV-labeled tracing showed that Spark-EVs were enriched with increased OVA antigens, DAMPs (e.g. HSPD1), which deliver into DCs then. Additionally, Integrated multi-level proteomics and prognostic analysis identified Junction plakoglobin (JUP) as a potential key transmitter of ICD signal. Conclusion : Using EV-based tracing, we visualized how LDRT triggers tumor cells to generate Spark-EVs, which deliver enhanced immunogenic signals to DCs. Furthermore, we delineate that radiation-responsive EV-borne proteins (e.g. JUP, HSPD1) that mediate the release, transmission, and effector functions of LDRT-induced immunogenic signaling. These findings establish Spark-EVs as key, targetable messengers of LDRT-driven ICD.