Fig 2: Anti-CSF-1R treatment promotes metastasis in the 4T1 model. (A) Growth curves of 4T1-luc mammary tumors treated with anti-CSF-1R (n = 10) or IgG control (n = 9) (both 200 µg/mouse every second day), two-way ANOVA. (B) On the day of sacrifice, the lymph nodes, lung and liver were dissected from the treated mice 10 min after a luciferin substrate injection and were imaged for metastatic burden using IVIS. The table shows metastasis positive organs on a yes/no basis, based on the IVIS signal and are shown as metastasis positive/total number of mice. The lower graphs show the radiance signal for each organ. Significant outliers were excluded from statistical analyses based on Grubbs' test and have been marked with a cross. Statistical significance was analyzed using two-tailed Student's t-test with Welch's correction. (C) ELISA analysis of serum M-CSF levels from mice treated either with IgG or anti-CSF-1R, two-tailed Student's t-test. (D) End point hematological analysis of monocytes analyzed with Sysmex XT-2000iV in IgG and anti-CSF-1R-treated mice. (E) Immunofluorescence staining of tumor sections (peritumoral area) with anti-F4/80 (green) and anti-MR antibodies (red) in IgG and anti-CSF-1R-treated mice. Quantification of double positive cells was done using ImageJ of three to five microscopic fields/mouse/treatment and analyzed using two-tailed Student's t-test. (F) Flow cytometric analysis of cell surface CSF-1R and G-CSFR expression on peripheral blood CD11b+Ly6C+ myeloid cells in C57BL/6 wild type (WT) and G-CSFR knockout (KO) mice where gate Q1 represents the monocytic population (Ly6G-) and gate Q3 the granulocytic population (Ly6G+). Because we used human recombinant G-CSF conjugated with AlexaFluor488 (G-CSF-488) for the detection of G-CSFR, the G-CSFR KO mice served as a control for specific binding of the protein. The histograms show binding of G-CSF-488 to the monocytic and granulocytic populations in WT (black line) and KO (gray-filled line) mice. (G) Flow cytometric analysis of cell surface CSF-1R and G-CSFR expression on peripheral blood CD11b+Ly6C+ myeloid cells in Balb/c mice without tumors or carrying a 4T1 tumor (size 5 × 5 mm). As in (F), the Q3 gate served as a positive cell population for G-CSFR expression, and is shown as the gray-filled histograms. Note that the 4T1 tumor induced a granulocytic cell population double positive for CSF-1R and G-CSFR (Q2).

Fig 3: Genetic and pharmacological inhibition of Apelin impairs mammary tumor growth and tumor angiogenesis in a paracrine manner Kaplan–Meier plot for survival in NeuT;Apln +/+ (n = 11) and NeuT;Apln -/- (n = 10) mice with mammary cancer after tumor onset. *P = 0.0185; log-rank test. Mice were sacrificed when the tumor size reached 1 cm3, following ethical guidelines.Tumor volumes, followed over time, of control mammary tumor E0771 cells (shRenilla) and Apelin-depleted (shApln) E0771 cells orthotopically injected into both syngeneic C57BL/6J Apln +/+ and Apln -/- mice (5 × 105 cells/mouse), respectively. Tumor volumes were determined using calipers and are shown as mean tumor volumes ± SEM. Data shown are pooled from two independent experiments. Apln +/+ ;shRenilla (n = 18), Apln +/+ ;shApln (n = 17), Apln -/- ;shRenilla (n = 14), Apln -/- ;shApln (n = 15); **P < 0.01, ***P < 0.001; two-way ANOVA.Mean percentages (±SEM) of CD31+ area in E0771 shRenilla (n = 3) or shApln (n = 3) mammary tumors, assessed on day 23 post-orthotopic injection into C57BL/6J Apln +/+ or Apln -/- mice, respectively. **P < 0.01; t-test.Mean percentages (±SEM) of extravasated Dextran in E0771 shRenilla (n = 9) or shApln (n = 12) mammary tumors, assessed on day 19 post-orthotopic injection into C57BL/6J Apln +/+ or Apln -/- mice, respectively. **P < 0.01; t-test. Right panel shows representative immunofluorescence of Dextran (red), CD31+ vessels (green), and DAPI (blue). The white arrows indicate regions of Dextran extravasation. Scale bars = 100 µm.Mean counts (±SEM) of pimonidazole positive foci, assessed on day 26 post-orthotopic injection of E0771 shRenilla (n = 6) or shApln (n = 4) into C57BL/6J wild-type mice (5 × 105 cells/mouse). *P < 0.05; t-test. Right panels show representative immunohistochemical pimonidazole staining at two different magnifications; scale bars = 200 µm (upper panels) and 50 µm (lower panels).Mean percentage (±SEM) of tumor-infiltrating immune cells normalized to CD45+. E0771 shRenilla (n = 8) or shApln (n = 6) were orthotopically injected into C57BL/6J Apln +/+ or Apln -/- mice (5 × 105 cells/mouse), respectively, and tumors were harvested day 25 post-injection. *P < 0.05, **P < 0.01; t-test. All immune cell populations were gated for viable CD45+ cells and then further defined as: CD8 T cells (Thy1.2+, CD8+), CD4 T cells (Thy1.2+, CD4+), inflammatory monocytes (Ly6C+, Cd11b+, Ly6G-), PMN-MDSCs (Ly6G+, Cd11b+), natural killer cells (Thy1.2-, Ly6G-, NK1.1+), natural killer T cells (Thy1.2+, CD4-, CD8-, NK1.1+), and peripheral dendritic cells (Ly6G-,Ly6C+, Cd11b-, PDAC1+, B220+). Source data are available online for this figure.

Fig 4: Representative Flow Cytometry Plots of Intratumorally Transferred Monocytes from Bone Marrow of LysMCre:Rosa26tdT:Zbtb46GFP MiceArrows: Plots before each arrow show the gating for cells shown on plots after the arrow. Cells were pregated on Ly6G- CD3e- NK1.1- live singlets and intratumorally transferred monocytes were subsequently identified as CD45+ tdT+.

Fig 5: P. aeruginosa upregulates CRAMP to promote NET formation via CLEC5A in vivo.(A) WT and Clec5a–/– mice were intratracheally inoculated with median lethal dose (LD50) of P. aeruginosa (Xen41, PAO1 strain), and BALF was harvested at 15 hours after infection to detect Ly6G+ cells (mock: WT, n = 6; Clec5a–/–, n = 3; WT + DNase 1, n = 4; Xen41LD50: WT, n = 7; Clec5a–/–, n = 6; WT + DNase 1, n = 8). (B) To detect NET formation, lung tissue was collected at 24 hours after infection and fixed in 10% formamide and embedded in paraffin. Tissue sections were stained with antibodies to myeloperoxidase (MPO) (green), citrullinated histone H3 (Cit-H3) (red), and Hoechst 33342 (blue) for NET structure visualization. Scale bar: 200 µm. (C and D) The areas of MPO (C) and MPO/Cit-H3 colocalization (D) were analyzed using MetaMorph software (n = 6 for each group). (E) Expression levels of Cramp mRNA in tissues were determined by qPCR (mock: WT, n = 4; Clec5a–/–, n = 3; Xen41LD50: WT, n = 4; Clec5a–/–, n = 4). (F) CRAMP protein in BALF was measured by ELISA (mock: WT, n = 5; Clec5a–/–, n = 3; Xen41LD50: WT, n = 8; Clec5a–/–, n = 7) (F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (2-way ANOVA). (G) Neutrophils from WT and Clec5a–/– mice were stimulated with CRAMP (50 µg/mL) at 37°C for 3 hours (mock: WT, n = 3; Clec5a–/–, n = 3; Xen41LD50: WT, n = 5; Clec5a–/–, n = 5). The level of NET formation was calculated from the area (µm2) of histone overlapping with MPO using MetaMorph software. Data are mean ± SEM from at least 3 independent experiments. The statistical significance was calculated with 2-way ANOVA for A and C and by unpaired and nonparametric Student’s t test with Mann-Whitney U test for D–G. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.