Murine dissociation kit

To obtain single cell suspensions, tumours were processed using a gentleMacs dissociator and a murine dissociation kit (Miltenyi Biotec, Surrey, UK). For staining of cells, non-specific binding was blocked with rat anti-CD16/CD32 Fc block on ice. Cells were incubated with Gr-1–FITC, CD11b–APC (eBioscience, Leicestershire, UK), washed in 1% FCS/PBS. For analysis, live cells were gated using vital dye exclusion (Invitrogen) and population phenotyped on FACs Canto (BD Bioscience) and analyzed using Flow Jo software. An example of the gating strategy employed is provided in Figure S5.

For flow cytometry analysis, tumors were dissociated with a gentle Macs Dissociator and a murine dissociation kit (Miltenyi Biotec, 130-096-730), as per manufacturing instructions. The dissociated cells were washed with 1% FBS in PBS buffer, and incubated on ice for 30 minutes with anti-CD16/CD32 Fc (ThermoFisher) to block any unspecific binding. Afterwards, cells were incubated in a master mix solution of 1% FBS in PBS buffer containing anti-CD45, anti-CD11b, anti-MHCII, anti-CD80, anti-CD86, anti-CD163 and anti-CD206. For analysis, live cells were gated using vital dye exclusion (L34963, ThermoFisher), and population phenotyped on Fortessa (BD) and analyzed via FlowJo software (version 10). Flow cytometry analysis for macrophages was performed on live single cells gated as CD45+CD11b+ cells, followed by the gating on each of the specific markers (i.e.: CD80, MHCII, CD86, CD206, CD163). M1 macrophages were defined as CD45+CD11b+CD80+ cells and as CD45+CD11b+MHCII+. M2 macrophages were defined as CD45+CD11b+CD86+, CD45+CD11b+CD206+, CD45+CD11b+CD163+ cells. Gates were determined by FMO controls for each specific marker.