Fig. 1

FAP⁺ HO-1⁺ TAMs in human and mouse breast adenocarcinoma are a tumour-educated phenotype. a, b Representative images of frozen human invasive ductal mammary carcinoma sections stained with DAPI (nuclei; blue) and antibodies against CD11b (green), FAP (yellow) and HO-1 (red) (representative images from n = 3 tumours). Arrows indicate cells co-expressing CD11b, FAP and HO-1. c, d Abundance (c) and surface characterisation (d) of live (7AAD⁻) CD45⁺ F4/80⁺ TAMs from enzyme-dispersed 4T1 tumours (at day 24 post inoculation) as assessed using flow cytometry. Each point in c represents live F4/80⁺ cells in an individual tumour. Histograms represent positive staining for the markers shown (blue shaded) against that of the respective isotype controls (grey shaded). e Flow cytometry gating strategy for live CD11b⁺ F4/80^low Ly6C^hi monocytes in a representative enzyme-dispersed 4T1 tumour; histogram shows surface FAP staining (red shaded) against that of the isotype control (grey shaded). f Median fluorescence intensity (MFI) of FAP surface expression with fluorescence minus one (FMO) background staining subtracted on CD45⁺ F4/80⁺ TAMs from enzyme-dispersed 4T1 tumours on the indicated day post injection of the tumour cells (n = 6). g, h Hmox1 mRNA expression relative to the housekeeping gene Tbp in 4T1 tumour tissue on the indicated day post inoculation of the tumour cells (n = 6–8 tumours per time point) (g) and FACS-sorted monocytes, FAP^lo F4/80^hi TAMs and FAP^hi F4/80^hi TAMs and 4T1 tumour cells (CD45⁻ Thy1.2⁻ CD31⁻) from duplicate wells from a 4T1 tumour, data representative of duplicate experiments (h). i Representative FAP staining of live (7AAD⁻) CD45⁺ F4/80^hi TAMs from an enzyme-dispersed E0771 tumour as assessed using flow cytometry. Histogram represents positive staining for FAP (black line) against isotype control staining (grey shaded). j Hmox1 mRNA expression relative to the housekeeping gene Tbp in FACS-sorted monocytes (CD11b⁺ F4/80^lo Ly6C^hi) and TAMs (F4/80^hi) from duplicate wells from an E0771 tumour, data representative of duplicate experiments. k, l 4T1 (blue) and E0771 (black open) tumours were grown concurrently in opposing mammary fat pads in Rag2^−/− mice. k Schematic representation of the experimental setting (top) and representative FAP expression as assessed using flow cytometry on live CD45⁺ F4/80^hi TAMs from enzyme-dispersed tumours (bottom). l Hmox1 mRNA expression relative to the housekeeping gene Tbp in MACS-sorted TAMs from enzyme-dispersed tumours (n = 4). Bar charts represent mean + s.d. *P < 0.05