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Background Peritoneal metastasis (PM) from gastrointestinal (GI) malignancies remains a major clinical hurdle, with no effective therapies. Appendiceal cancer, though rare and increasingly diagnosed, often resists colorectal cancer (CRC) chemotherapy, subjecting patients to toxicity without benefit. The peritoneal fluid is enriched with immune cells—macrophages, dendritic cells, and T cells—implicated in metastatic progression, yet their phenotypic diversity across GI PM subtypes and roles in therapy response are poorly defined. A clinical trial ( NCT06207305) led by Drs. Helmink and Shen evaluates the safety and efficacy of intraperitoneal (IP) paclitaxel (PTX) treatment for appendiceal PM patients. Here, we characterize the peritoneal immune cell dynamics in individuals without PM versus those with CRC- or appendiceal-derived PM and assess how IP chemotherapy shapes immune phenotypes and influences treatment outcomes.Methods Single-cell RNA sequencing and single-cell T cell receptor sequencing were performed on peritoneal fluid cells isolated from eight CRC PM samples, ten appendiceal PM samples from six patients—including four pre- and post-IP PTX treatment pairs—and seven healthy controls. scRNA-seq data were processed using Seurat, with Harmony integration applied as needed to mitigate batch effects. To assess the functional role of peritoneal macrophages in GI PM progression and IP PTX efficacy, 500,000 MC38-luciferase cells were injected intraperitoneally into wild-type or Lyz2Cre-Abca1/Abcg1 flox/flox mice, followed by weekly IP PTX or vehicle. Tumor burden was quantified using the IVIS50 bioluminescence imaging system.Results Overall immune profiling revealed comparable macrophage and DC abundance across groups, but notably reduced T cell proportions specifically in appendiceal PM patients. Peritoneal macrophages from PM patients displayed enriched efferocytosis and lipid metabolism pathways, indicating potential for tumor cell engulfment. Most macrophage subsets, including tumor-associated macrophages (LYVE1+SPP1+), were similarly prevalent in both healthy and cancerous peritoneal fluid, suggesting a baseline anti-inflammatory peritoneal environment. IP PTX treatment markedly shifted macrophage composition toward subsets enriched in efferocytosis, cholesterol metabolism (ABCA1, ABCG1), and multidrug resistance pathways (ABCB1, ABCC1). Pathway analysis indicated that macrophages post-treatment exhibited enriched pro-inflammatory signatures, such as IFNA and IFNB signaling pathways, suggesting chemotherapy resistance combined with pro-inflammatory features. In DC2s, IP PTX induced upregulation of antigen-processing, costimulatory molecules, cytokine/chemokine signaling, and migratory pathways, potentially enhancing T cell-mediated antitumor responses. In a mouse model, macrophage-specific knockout of Abca1/Abcg1 increased tumor burden and impaired IP PTX efficacy.Conclusions These results demonstrated that IP paclitaxel reprograms peritoneal macrophages and DCs toward pro-inflammatory and immunostimulatory phenotypes, underscoring the potential roles of MNPs in determining chemotherapy efficacy.Acknowledgements This work was supported by funding from the American Surgical Association Research Award (Dr. Beth A. Helmink), NIH grant R37AI049653 (Dr. Gwendalyn J. Randolph), and NIH grant K00CA264434 (Dr. Jichang Han). We gratefully acknowledge all patients who generously contributed clinical samples and participated in this study.Trial Registration NCT06207305Ethics Approval Patients were recruited from the Barnes-Jewish Hospital and the University of Texas MD Anderson Cancer Center, USA. All human studies were performed in accordance with ethical regulation and preapproved by the Washington University Human Research Protection Office, the MD Anderson Cancer Center Office of Human Subject Protection, the Washington University Institutional Review Board (#201903051) and the MD Anderson Cancer Center Institutional review Board (#IRB00000121)Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal