Fig 2: Increased blood neutrophils and AnxA1 levels in the lung melanoma metastasis model. (A) Amounts of neutrophils in the peripheral blood from lung melanoma metastasis mice. (B) Serum levels of AnxA1. (C) Secretion of AnxA1 by melanoma cells (B16F10) and neutrophils cultured for 24 h. The data represent the average ± SEM. * p < 0.05 vs. control; *** p < 0.001 vs. B16F10. Control group (n = 5); melanoma group (n = 5).

Fig 3: Neutrophil depletion reduced the number of melanoma lung metastasis and serum AnxA1 levels. (A) Experimental design. (a) Total number of neutrophils and monocytes in peripheral blood. (B,C) Images of the lungs from isotype or anti-Gr1-treated mice. (D) Number of melanoma metastasis spots. (E,F) Morphologic analysis by H&E staining. (G,H) Representative images of the Melan A expression (I,i) in isotype and (J,j) neutrophil-depleted mice. Insert: Melan A expressed in cells spread into the lungs. Red arrows: immune cells labeled for AnxA1. (K) Serum levels of AnxA1. The data represented the average ± SEM. * p < 0.05 vs. isotype. Melanoma group (n = 4, 5). Melanoma neutrophil-depleted group (n = 5).

Fig 4: Increased expression and serum levels of AnxA1 in lung melanoma metastasis mice. (A) Experimental design. (B,C) Control group. Alveolar epithelium (arrowhead). Bronchiolar epithelium (Br). (D,E) Expression of AnxA1 in melanoma cells (T) from lung melanoma metastasis. (F,G) Immune cells labeled for AnxA1. Immune cells infiltrate (arrows). Inserts: Tumor and immune cells. Ly6G-positive cells in (H) control and (I) melanoma groups. (J) Increased number of neutrophils in lung melanoma metastasis mice. (K) AnxA1 expression in neutrophils (Ly6G+) and (L) in melanoma cells from lung melanoma metastasis. (M) Amounts of AnxA1 expression in neutrophils and melanoma cells. The data represent the average ± SEM of at least 3–5 animals per group. p * < 0.05 vs. control; ** p < 0.01 vs. neutrophils.

Fig 5: Expression and serum levels of AnxA1 were increased in melanoma patient samples. (A–C) Representative image of inflammatory infiltrate in biopsies obtained from melanoma patients—H&E staining (black arrows and inserts: lymphocytes). Scale bar: 100 µm. (D) H&E staining and (E) AnxA1 expression detected by immunofluorescence in nevus sample. (F,H,J) H&E staining and (G,I,K) AnxA1 expression detected by immunofluorescence in melanoma cells from melanoma biopsies. (*) pigmented cells. (Black arrows) melanoma cells. (White arrows) AnxA1-labeled cells. Scale bar: 50 µm. (L) Leukogram. (M) AnxA1 expression in circulating neutrophils (CD66+) evaluated by flow cytometer. (N) AnxA1 serum levels. The data represent the average ± SEM. Control (n = 4); nevus (n = 4–5); melanoma (n = 5–9). * p < 0.05 vs. nevus; ## p < 0.01 vs. control.