Exosome whisperers: Decoding SPHK1's role in cisplatin-induced immune escape of NSCLC.

Abstract

e20622 Background: Cisplatin, a key NSCLC chemotherapy, exhibits both cytotoxic and immunomodulatory effects. It influences tumor-derived exosomes (TDEs), critical carriers of bioactive molecules that reshape the tumor immune microenvironment (TIME) and promote immune evasion. While cisplatin is known to alter TDE secretion, contributing to an immunosuppressive TIME, the specific molecular mechanisms and functional consequences remain unclear. This study aims to elucidate how cisplatin reprograms exosomal cargo and its subsequent impact on tumor progression and immune escape in NSCLC. Methods: Transcriptome sequencing analyzed cisplatin's impact on RNA profiles in serum exosomes from NSCLC patients. Exosome biogenesis and secretion were assessed via immunofluorescence and automated fluorescence detection. RNA pull-down with mass spectrometry identified key mechanisms for SPHK1 mRNA loading into exosomes. Cisplatin's effect on the SPHK1/S1P pathway was evaluated by Western blot, qPCR, and ELISA, while ChIP-qPCR and dual-luciferase assays verified E2F1-mediated transcriptional regulation. SPHK1's role in proliferation and PD-L1 expression was tested via proliferation assays and Western blot. Exosome uptake was observed using FISH and immunofluorescence; flow cytometry and immunofluorescence assessed recipient cell changes. An orthotopic lung cancer mouse model treated with exosomes underwent single-cell sequencing to evaluate tumor immune microenvironment alterations. Results: Cisplatin induces dual pro-tumorigenic effects in NSCLC: it enhances exosome biogenesis and promotes STAU1-mediated sorting of SPHK1 mRNA into these vesicles. Concurrently, cisplatin transcriptionally upregulates SPHK1 via the transcription factor E2F1. Exosomes enriched with SPHK1 mRNA are internalized by recipient cancer cells, driving proliferation and upregulating PD-L1 expression. In a lung cancer mouse model, treatment with exosomes from SPHK1-knockdown cells (sh exo) inhibited tumor growth and remodeled the tumor immune microenvironment. Single-cell analysis revealed sh exos increased cytotoxic CD8⁺ T and NK cell infiltration while reducing Tregs and exhausted T cells, accompanied by enhanced expression of T-cell recruitment (CXCR6), cytokine signaling (Il2rg), and cytotoxic effector molecules (IFN-γ, GzmB, Pfn1). Conclusions: Cisplatin simultaneously triggers an exosome-mediated signaling axis that may counteract its efficacy by promoting tumor growth and fostering an immunosuppressive microenvironment through PD-L1 induction.