Extended Data Figure 9: Analysis of Alox5-null bone marrow chimaeric mice transplanted with primary mammary MMTV-PyMT tumours and failure of Alox5-null neutrophils to support cancer cell metastatic initiation potential.
From: Neutrophils support lung colonization of metastasis-initiating breast cancer cells
a, Efficiency of chimaeric mice generation was determined by semi-quantitative PCR analysis of DNA isolated from the bone marrow of lethally irradiated wild-type mice reconstituted with wild-type or Alox5-null bone marrow. A calibration curve of the ratio between the PCR band amplified from the wild-type (WT) and Alox5-null (KO) allele was used to calculate the percentage of bone marrow reconstitution efficiency. Tests of 8 representative Alox5−/− chimaeric mice and 10 controls are shown. Only mice with >80% Alox5-null bone marrow reconstitution were used for further experiments. b–d, Analysis of wild-type and Alox5-null bone marrow chimaeric mice 1.5 months after transplantation with 2 mammary MMTV-PyMT tumours (106 PyMT cells) or tumour-free controls. Representative flow cytometric analysis (b) and quantification of CD11b+Ly6G+ neutrophil presence in the lung (c) (n = 4 (wild type), n = 4 (Alox5−/−), n = 5 (wild-type PyMT), n = 7 (Alox5−/− PyMT) as well as primary mammary tumour burden (n = 6 (wild-type PyMT), n = 9 (Alox5−/− PyMT)) (d). e, f, 5 × 105 luciferase-expressing MMTV-PyMT cells treated with control, wild-type LuN (LuN-WT) or Alox5-deficient neutrophil-derived LuN (LuN-Alox5ko) medium for 3 days in adherent culture were intravenously injected into Rag1-null mice. Quantification of cancer-cell-derived bioluminescence in the lung over time (n = 5 (control), n = 5 (LuN-WT), n = 4 (LuN-Alox5ko)) (e) and representative image is shown (f). Statistical analysis by two-sided t-test. Data are represented as mean ± s.e.m. *P < 0.05, **P < 0.01. Blot source data are shown in Supplementary Fig. 2.
