Figure. 3. Restoring extracellular ELANE activity in murine neutrophils attenuates tumorigenesis.

(A) Effect of CTSC, pro-mELANE, or mELANE (3μg/mL, 24h) on cancer and non-cancer cell viability (calcein-AM). n=6/group.

(B) ELANE catalytic activity in murine and human neutrophil media. n=4/group. Immunoblotting detects mELANE in murine neutrophil media (inset).

(C) Comparison of ELANE catalytic activity in murine neutrophil media (n=4/group) and lysates (n=2/group) isolated from various sources.

(D) Serine protease inhibitor levels in human PMN and murine PN media quantified by shotgun proteomics, n=2/group. See also Table S3.

(E) Immunoblots of SLPI in human and murine neutrophil media. Recombinant human SLPI (rhSLPI) was included as a control.

(F) Co-immunoprecipitation of SLPI with mELANE in murine PN media.


(G) mELANE activity in PN media from Slpi+/+, Slpi−/− mice with or without PMSF treatment, or Slpi−/− Elane−/− mice (left). Effects of those media on MDA-MB-231 cell viability (calcein-AM) (right). n=3/group.

(H-J) B16F10 lung tumor colonization in whole animal Slpi−/− mice and Slpi+/+ control mice (left), along with representative images (right); n=6–10/group (H). Effect of neutrophil depletion (I) on lung tumor colonization in Slpi+/+ and Slpi−/− mice (J); n=7–11/group.

(K) Schematic for generating Slpi+/+ or Slpi−/− chimeric mice (top). Engraftment efficiency was quantified using defined mixtures of Slpi+/+ and Slpi−/− bone marrow cells (bottom).

(L) B16F10 lung tumor colonization in Slpi+/+ and Slpi−/− chimeric mice; n=11/group.

*, p<0.05 Student’s t-test, data are mean ± SEM.