Fig 1: Osteosarcoma induces neutrophil extracellular traps (NETs) to promote OS progression. (A) Immunofluorescence staining results showed neutrophil extracellular traps in osteosarcoma tissues (green: Histone H3; red: ELA2; blue: DAPI; scale bar: 5 μm). (B) NET concentrations were tested in neutrophils, coculture system, and coculture with GSK484 by ELISA assay (one-way ANOVA test, N = 3 for each group). (C) The results of OS cell viability in the Ctrl group, neutrophil conditional medium group (NEU-CM), cocultured system conditional medium (co-CM), and cocultured system with GSK484 conditional medium (co-CM+GSK484) (one-way ANOVA test, N = 3 for each group). (D) ELA2 expression was tested in osteosarcoma cells, neutrophils, and a coculture system by ELISA assay (one-way ANOVA test, N = 3 for each group). (E) The schematic diagram of constructing tumor-bearing Balb/c mice and anti-ly6G intraperitoneal injection administration to exhaust neutrophils. (F) After anti-ly6G administration, IHC staining results indicated neutrophils were obviously decreased (N = 4 for each group; scale bar: 100 μm). (G) Images of osteosarcoma tissues from the anti-IgG group and the anti-Ly6G group. (H) Tumor weight and tumor volume were measured and quantitatively analyzed (tumor volume = length × width2/2; unpaired two-tailed Student’s t-test, N = 4 for each group). (I) Results of ELA2 IHC staining showed that anti-ly6G significantly inhibited ELA2 expression (scale bar: 100 μm). (J) The schematic diagram of our research shows that osteosarcoma induced neutrophil extracellular trap formation to secrete ELA2, which was taken up by osteosarcoma and promoted LMW-cyclin E1 formation, accelerating tumor progression (**P<0.01, ***P<0.001, ****P<0.0001).
Fig 2: Neutrophils released ELA2 to induce LMW-cyclin E1 formation. (A) The expression of ELA2 and the infiltration of neutrophils in primary osteosarcoma tissues (N = 34) and lung metastasis tissues (N = 33) were assessed using IHC staining. The IHC scores of ELA2 and CD66b in the primary osteosarcoma group and the lung metastasis group are shown. Positive neutrophil infiltration was correlated with osteosarcoma lung metastasis (Fisher’s exact test; scale bar: 100 μm). (B) IHC staining images of CD66b and ELA2 at the same location in two patients. The analysis of the relationship between neutrophils and ELA2 revealed that greater neutrophil infiltration indicated higher ELA2 expression (Chi-square test; scale bar: 100 μm). (C) Flow cytometry was used to examine the purity of isolated neutrophils, with CD66b+ cells gated as neutrophils. (D) Giemsa staining was performed to observe the morphology of the isolated neutrophils, which exhibited multilobed, dark purple nuclei (scale bar: 15 μm). (E) Schematic diagram illustrating the contact coculture and noncontact coculture systems. (F) Western blot analysis of cyclin E1 (FL, LMW) and ELA2 expression after neutrophil contact or noncontact coculture with osteosarcoma (the assay was replicated three times). (G) The results showed that the expressions of ELA2 and LMW-cyclin E in osteosarcoma were upregulated and increased downstream RB phosphorylation by neutrophils. AZD9668 could reverse the ELA2 effect on osteosarcoma (the assay was replicated three times). (* p < 0.05).
Fig 3: Calpain 1 and calpain 2 regulated FL-cyclin E1 expression, and neutrophil elastase (ELA2) accelerated LMW-cyclin E1 formation. (A) Western blot analysis of cyclin E1 (FL and LMW), calpain 1, calpain 2, and ELA2 expression in six OS cell lines. GAPDH served as the loading control (the assay was replicated three times). (B) Quantitative analysis of the relative mRNA expression levels of ELANE, CAPN1, and CAPN2 in OS cell lines by qPCR assay. (C) The interaction relationships between cyclin E1 and ELA2, and calpain 1 and calpain 2 were examined by IP–Western blot assays in U2OS and 143B (the assay was replicated three times). (D) Immunofluorescence staining showed that ELA2 was expressed in U2OS and 143B cell lines and could be secreted outside the cells (green: ELA2; red: F-actin; blue: DAPI; scale bar: 20 μm). (E) The protein expression of cyclin E1 (FL and LMW) and its downstream proteins (RB and p-RB) was assessed by Western blot after ELA2-OE plasmid transfection in U2OS and 143B cells (the assay was replicated three times). (F) Immunofluorescence images showed cyclin E1 expression after ELA2-OE plasmid transfection. Increased ELA2 upregulated cellular cytoplasmic cyclin E (Rred: cyclin E1; blue: DAPI; scale bar: 100 μm). (FL, full length; LMW, low molecular weight; p-RB, phosphorylated RB).
Fig 4: Osteosarcoma neutrophil elastase (ELA2) promotes osteosarcoma cell proliferation in vitro and in vivo. (A) Colony formation assay showed that ELA2 accelerated osteosarcoma proliferation in U2OS and 143B cells (unpaired two-tailed Student’s t-test, N = 3). (B) Immunofluorescence image showed that Lv-ELA2 was successfully transfected into K7M2 cell lines and stably expressed (scale bar: 100 μm). (C) Quantitative analysis of relative ELA2 expression level in the Lv-Ctrl group and Lv-ELA2 group by qPCR assays (unpaired two-tailed Student’s t-test, N = 3). (D) The results of the cell cycle in the Ctrl group and ELA2-OE group using flow cytometry (left). The statistical analysis of G0/G1 phase, S phase, and G2/M phase cell proportions (right) (unpaired two-tailed Student’s t-test, N = 3). (E) Images of tumors from the Ctrl group and ELA2-OE group tumors (N = 3 for each group). (F) Tumor weight and tumor volume were measured and quantitatively analyzed (tumor volume = length × width2/2; unpaired two-tailed Student’s t-test, N = 3). (G) Images of Ki-67+ immunohistochemistry staining in the Ctrl group and ELA2-OE group (scale bar: 100 μm). (*** p < 0.001).
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