SiglecF ^hi TANs mediate their protumorigenic functions via TGFβ secretion. (A–C) SiglecF^hi TANs were co-cultured with RIL175 cells, with or without the addition of Vactosertib (10 nM). In vitro limiting dilution (A), clonogenic (B), and scratch wound (C) assays were performed. (D–G) NRAS/AKT HCC tumors were induced in Mrp8^Cre+ve.TGFβ^fl/fl mice (Mrp8^Cre.TGFβ^fl/fl) and Mrp8^Cre−ve.TGFβ^fl/fl littermates (W/T). Representative FACs plots and percentage of Ki-67⁺ tumor cells (D). Representative FACs plots and percentage of CD13⁺CD133⁺ CSCs (E). Representative images of HCC livers and liver-to-body weight ratio. Scale bar represents 1 cm (F). Representative images of AFP⁺ tumors and tumor nodule counts. Scale bar represents 100 μm (G). (H–J) SiglecF^– and SiglecF^hi TANs were sort-purified from NRAS/AKT HCC tumors and subcutaneously injected alongside RIL175_shTgfbr1 cells into C57BL/6J mice. Schematic diagram of adoptive transfer strategy (H). Representative images and volumes of tumors at the endpoint. Scale bar represents 1 cm (I). Representative FACs plots and percentage of Ki-67⁺ tumor cells (J). Each symbol represents TANs isolated from two to three mice (B and C), or one mouse (D–G, I, and J). Data are mean ± SEM and represent two to three independent experiments (A, C–G, I, and J) or are pooled from two independent experiments (B). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Pearson’s chi-square test with a 95% confidence interval (A), one-way ANOVA (B and C), or unpaired Student’s t test (D–G).