A phase I window-of-opportunity trial of intraperitoneal, intratumoral lipopolysaccharide in peritoneal metastases: Safety and immune remodeling from the RIOT-1 study.

2603 Background: Peritoneal metastases (PM) from gastrointestinal malignancies exhibit immune exclusion and limited responsiveness to systemic immunotherapy. Regional intratumoral immune activation may reprogram the tumor microenvironment (TME). RIOT-1 evaluated the safety, feasibility, and biological effects of intraperitoneal intratumoral lipopolysaccharide (LPS), a Toll-Like Receptor-4 (TLR4) agonist, administered during diagnostic laparoscopy. Methods: RIOT-1 (NCT05751837) was a single-center, open-label Phase I window-of-opportunity trial. Twelve patients with appendiceal or colorectal PM received paired intratumoral injections of 1 µg E. coli O113-derived LPS and normal saline into spatially distinct tumor deposits at laparoscopy, followed by planned cytoreductive surgery 14 days later. The primary endpoint was safety. Secondary endpoints included immune and molecular remodeling assessed by immunohistochemistry (IHC), PhenoCycler multiplex immunofluorescence, spatial neighborhood analysis (CytoMAP), and high-resolution mass-spectrometry proteomics. Key proteomic findings were validated by targeted IHC staining. Results: Intratumoral LPS administration was feasible and well tolerated, with no grade ≥3 adverse events and no interference with subsequent cytoreductive surgery. IHC demonstrated significant post-LPS increases in M1-like macrophages (CD68⁺CD86⁺), M2-like macrophages (CD68⁺CD206⁺), and CD1a⁺ antigen-presenting cells (all p<0.05). PhenoCycler and CytoMAP analyses revealed LPS-specific reorganization of the immune microenvironment, characterized by enrichment of myeloid- and APC-dominant neighborhoods and altered tumor–immune spatial relationships compared with saline-injected controls. Proteomic profiling quantified 5,178 proteins and revealed LPS-specific enrichment of neutrophil degranulation, cytokine-mediated signaling, RNA translation, and autophagy–lysosomal pathways, distinct from biopsy/carrier-associated structural remodeling observed with saline. Upregulated proteins including LYZ, FCER1G, NAIP, and CD68 were confirmed by IHC, supporting localized innate immune activation and metabolic reprogramming. Conclusions: Intraperitoneal intratumoral LPS delivery in PM is safe and biologically active, inducing reproducible innate-dominant immune and metabolic remodeling validated across spatial, proteomic, and histologic platforms. These data establish clinical proof-of-mechanism for regional TLR4 agonism and support further dose-optimization and rational combination strategies in peritoneal malignancies. Clinical trial information: NCT05751837 .