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. 2022 Nov 15;132(22):e153643. doi: 10.1172/JCI153643

Figure 5. Tumor-derived ANXA2 drives ARG1 gene expression in tumor-associated neutrophils.

Figure 5

Arg1 mRNA expression in PMNs from the BM, spleen (Spl), tumor (TANs), HDNs, and LDNs from (A) PL-tumor-bearing (BM, n = 25; Spl, n = 28; TAN, n = 28; HDN, n = 5; LDN, n = 5) and (B) LLC-tumor-bearing mice (BM, n = 3; Spl, n = 4; TAN, HDN, LDN, n = 5). (C) C57BL/6 BM PMNs treated with PBS (n = 7), PL tumor lysate (PL-TL, 100 μg/mL, n = 9), heat-shocked PL-TL (100 μg/mL, n = 6) or adjacent lung lysate (100 μg/mL, n = 3) for 16 hours. Data expressed as fold change in Arg1 mRNA expression compared with PBS control. (D) C57BL/6 HDNs treated with PBS (n = 5), PL-TL (100 μg/mL, n = 11), and LLC-TL (100 μg/mL, n = 4) for 16 hours. Data expressed as fold change in Arg1 mRNA expression compared with PBS control. C57BL/6 BM PMNs treated with equal volume (100 μL) of FPLC fractions from (E) PL-TL or (F) LLC-TL for 16 hours. Data expressed as fold change in Arg1 mRNA expression compared with PBS control (n = 3). (E and F) Representative Western blot for ANXA2 in FPLC fractions 2, 3, and 20 from PL-TL and LLC-TL, respectively. C57BL/6 HDNs treated with (G) PL-TL (100 μg/mL) and (H) LLC-TL (100 μg/mL) in the presence of control peptide (LGKLSK, 10 μM) or ANXA2-blocking peptide (LCKLSK, 10 μM) for 16 hours. Data expressed as fold change in ARG1 gene expression compared with PBS control. (I) BM PMNs (n = 3) and (J) HDNs (n = 4) isolated from C57BL/6 mice treated with recombinant mouse ANXA2 (rANXA2; 1 μg/mL) for 16 hours. Data expressed as fold change in Arg1 mRNA expression compared with PBS control. (K) CM from HDNs isolated and treated as in J and analyzed by ARG1 ELISA (control, n = 5; rANXA2, n = 7). Data expressed as ARG1 protein in pg/mL. (L) ARG1 activity assay present in the CM for WT and Arg1–/– PMNs incubated with vehicle, PL-TL, or rANXA2. n = 5 per group. *P < 0.05 compared with control. (M) IFN-γ activity assay for column-purified splenic CD8+ T cells (n = 5), with anti-CD3/anti-CD28 activation (1 μg/mL) (n = 5), and coculture with WT (n = 7) or Arg1–/– (n = 4) PMNs at a 1:1 ratio. (N) Representative images for FFPE slides from PL and LLC tumors (n = 4 each) stained for ANXA2 (purple), F4/80 (yellow), CK (white), and with DAPI (blue). *P < 0.05 by 1-way ANOVA with Tukey’s post hoc test (A–D, G, H, L, and M) or 2-tailed Student’s t test (I).