Figure S4.
Identification of factors contributing to the development of SiglecFhiTANs. (A) Quantities of SiglecF− and SiglecFhi TANs after overnight culture with or without 100 μM 10058-F4. (B) Representative immunofluorescence image and proximity of TAN subsets to lipid droplets (MPO: red; SiglecF: green; BODIPY 493/503: pink; nucleus: blue) in NRAS/AKT HCC tumors. Scale bar represents 10 µm. (C–E) BM neutrophils were isolated from NRAS/AKT HCC-bearing mice and cultured overnight with or without the addition of the indicated fatty acids (50 μM). Representative FACs plots of SiglecF+ neutrophils (C). Representative histograms of c-Myc expression (D). TGFβ1 production measured by ELISA following 4 h of LPS (100 ng/ml) stimulation (E). (F) Expression of Csf2 by cellular populations within NRAS/AKT HCC tumors. (G) NRAS/AKT HCC was induced in C57BL/6J or GM-CSF reporter Csf2-iCre-EGFP+/wt mice. Representative FACs plots of GM-CSF producing cells. (H) Representative FACs plots of SiglecF+ neutrophils after 20 ng/ml GM-CSF was included in C. (I and J) Neutrophils from spleen and tumor of NRAS/AKT mice were cultured overnight with or without the addition of GM-CSF (20 ng/ml) or/and linoleic acid (50 μM). c-Myc expression within splenic neutrophils (I) or SiglecF− and SiglecFhi TANs (J). (K) Pseudotime of TAN development in NRAS/AKT HCC mice, calculated with Monocle 3. (L) SiglecF− or SiglecFhi TANs isolated from CD45.1+ mice were adoptively transferred into NRAS/AKT HCC-bearing CD45.2+ recipients. After 2 days, TILs were harvested, donor TANs (CD45.1+, polygon gate) and endogenous TANs were assessed for their SiglecF expression (rectangle gate indicates SiglecFhi cells). (M and N) BM neutrophils were isolated from naïve mice and cultured with and without GM-CSF (20 ng/ml) and linoleic acid (50 μM) overnight. Representative histograms of c-Myc expression (M). TGFβ1 production measured by ELISA following 4 h of LPS (100 ng/ml) stimulation (N). Each symbol represents TANs isolated from two to three mice (A and J), or one mouse (E, I, and N), or one neutrophil from screening liver sections of eight mice (B). Data are mean ± SEM and are representative of two independent experiments (C–E, G, H, and L–N) or are pooled from two independent experiments (A, B, I, and J). *P < 0.05 by unpaired Student’s t test (B). CM, complete media.

Identification of factors contributing to the development of SiglecF hi TANs. (A) Quantities of SiglecF− and SiglecFhi TANs after overnight culture with or without 100 μM 10058-F4. (B) Representative immunofluorescence image and proximity of TAN subsets to lipid droplets (MPO: red; SiglecF: green; BODIPY 493/503: pink; nucleus: blue) in NRAS/AKT HCC tumors. Scale bar represents 10 µm. (C–E) BM neutrophils were isolated from NRAS/AKT HCC-bearing mice and cultured overnight with or without the addition of the indicated fatty acids (50 μM). Representative FACs plots of SiglecF+ neutrophils (C). Representative histograms of c-Myc expression (D). TGFβ1 production measured by ELISA following 4 h of LPS (100 ng/ml) stimulation (E). (F) Expression of Csf2 by cellular populations within NRAS/AKT HCC tumors. (G) NRAS/AKT HCC was induced in C57BL/6J or GM-CSF reporter Csf2-iCre-EGFP+/wt mice. Representative FACs plots of GM-CSF producing cells. (H) Representative FACs plots of SiglecF+ neutrophils after 20 ng/ml GM-CSF was included in C. (I and J) Neutrophils from spleen and tumor of NRAS/AKT mice were cultured overnight with or without the addition of GM-CSF (20 ng/ml) or/and linoleic acid (50 μM). c-Myc expression within splenic neutrophils (I) or SiglecF− and SiglecFhi TANs (J). (K) Pseudotime of TAN development in NRAS/AKT HCC mice, calculated with Monocle 3. (L) SiglecF− or SiglecFhi TANs isolated from CD45.1+ mice were adoptively transferred into NRAS/AKT HCC-bearing CD45.2+ recipients. After 2 days, TILs were harvested, donor TANs (CD45.1+, polygon gate) and endogenous TANs were assessed for their SiglecF expression (rectangle gate indicates SiglecFhi cells). (M and N) BM neutrophils were isolated from naïve mice and cultured with and without GM-CSF (20 ng/ml) and linoleic acid (50 μM) overnight. Representative histograms of c-Myc expression (M). TGFβ1 production measured by ELISA following 4 h of LPS (100 ng/ml) stimulation (N). Each symbol represents TANs isolated from two to three mice (A and J), or one mouse (E, I, and N), or one neutrophil from screening liver sections of eight mice (B). Data are mean ± SEM and are representative of two independent experiments (C–E, G, H, and L–N) or are pooled from two independent experiments (A, B, I, and J). *P < 0.05 by unpaired Student’s t test (B). CM, complete media.

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