Extended Data Figure 6. Neutrophil isolation from the lung of MMTVPyMT+ mice and effect of neutrophil-derived factors on tumour formation potential.
a, Representative flow cytometric analysis of neutrophil purity after isolation from the pre-metastatic lung compared to total lung tissue. Only neutrophil purity of ≥90% was used for further experiments. b, Neutrophil viability was assessed by flow cytometry for propidium iodide (PI) negativity after isolation (n = 10). c, d, MMTVPyMT cells grown in control or LuN medium for 3 days in adherent conditions were plated in non-attachment conditions followed by sphere quantification at day 10 post-seeding (technical replicate n = 17 (control), n = 21 (LuN) of biological triplicates) (c) or 104 cells grafted onto the mammary gland of Rag1-null mice for analysis of tumour formation potential (d). Tumour burden was determined by weighing about 3 weeks after (n = 12 per group), complementary to Fig. 2d. e–h, Flow cytometric quantification of frequencies of total present GFP-labelled MMTV-PyMT cells (e, g) and frequencies of CD24+CD90+ MICs among total GFP-labelled MMTV-PyMT cells (f, h) in the lung of Rag1-null mice 3 days after intravenous injection of 5×105 total GFP-labelled MMTV-PyMT cells followed by either three intravenous injections with control or LuN medium (n = 6 (PyMT+control), n = 8 (PyMT+LuN)) (e, f) or by one intravenous injection with 25×106 neutrophils freshly isolated from a pre-metastatic lung (n = 7 (PyMT control), n = 8 (PyMT+neutrophils) (g, h). f, h, Two independent experiments are shown to complement Fig. 2h, i. Exp, experiment. i–k, Experimental setup (i): Rag1-null mice were intravenously injected with 1–10×105 (j) or 0.5×106 total GFP-labelled MMTV-PyMT cells (k) followed by either 3-5 intravenous injections with 200μl control or LuN medium (j) or by three intravenous injections with 25×106 neutrophils (k) freshly isolated from a pre-metastatic lung. Quantification of experimental metastatic incidence by determination of bioluminescence intensity (n = 7 (control), n = 9 (LuN)) (j) or flow cytometric analysis of GFP+ cancer cells in the lung (n = 5 (control), n = 4 (neutrophil)) (k) is shown. Statistical analysis by two-sided t-test (c, j, k) and two-way ANOVA (d–h). Data are represented as mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.