Association of IL4I1+ M2-like macrophages in tumor microenvironment with poor prognosis in hepatocellular carcinoma: insights from bioinformatics and experimental validation

Background Hepatocellular carcinoma (HCC) is associated with poor prognosis and limited responses to immunotherapy, partly due to the immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages, especially M2-like macrophages, play important roles in HCC progression. Interleukin-4-induced gene 1 (IL4I1), a tryptophan-metabolizing enzyme, has been implicated in tumor immune regulation. However, the role and prognostic significance of IL4I1-expressing M2-like macrophages in HCC remain unclear. This study investigated the expression pattern, spatial distribution, functional association, and prognostic relevance of IL4I1-expressing M2-like macrophages in HCC and their association with patient outcomes. Methods We analyzed public databases (TIMER, UALCAN, TISCH2) for gene expression and prognostic significance. An in vitro co-culture system was established using a Huh7 HCC cell line and THP-1-derived M2-like macrophages with and without IL4I1 knockout via CRISPR/Cas9. We assessed cell proliferation, migration, apoptosis, and cytokine profiles. Multiplex immunofluorescence (mIF) was performed on tissue microarrays from 92 HCC patients to analyze the spatial distribution of IL4I1+ M2-like macrophages. Results IL4I1 was highly expressed in HCC, predominantly within macrophages, and correlated with poor prognosis. In a THP-1-derived macrophage/Huh7 co-culture model, IL4I1 expression in M2-like macrophages was associated with increased Huh7 cell proliferation and migration and reduced apoptosis. This was associated with increased secretion of pro-inflammatory cytokines (e.g., CCL15, TNF-α) and decreased levels of IL-10 and TGF-β1. mIF analysis further showed that a high density of IL4I1+ M2-like macrophages in the epithelial/parenchymal tumor region was significantly associated with poorer overall survival ( p = 0.033). Conclusions IL4I1+ M2-like macrophages are associated with a pro-tumorigenic microenvironment and adverse patient outcomes in HCC. Our findings support IL4I1+ M2-like macrophages as a prognostically relevant macrophage subset, while further mechanistic validation, particularly of IL4I1-derived metabolites and AHR pathway activation, is required before IL4I1 can be considered a validated therapeutic target.