Tumor and Splenic Immune Cell Profiling

The tumour flow cytometry-based immuno phenotype was performed according to already published protocols^{45 (link)}. Briefly, tumours were minced and digested for about 1 hr at 37 °C under continuous mixing with a digestive mix containing 1 mg/mL collagenase IV, 0.1 mg/mL hyaluronidase, and 30 U/mL DNAse in RPMI 1640, all purchased from Sigma-Aldrich. The cell suspension was separated from the undigested material using a 70-μm cell strainer (Corning). On the contrary, the analysis of circulating leukocytes was performed using splenocytes collected by mechanical disruption of the tissue followed by red blood cell lysis with the ACK buffer (Lonza). One million of cells were incubated with anti-mouse CD16/32 (Biolegend) and subsequently stained with the following antibodies according to the vendor’s instructions: CD3 (17A2, Thermo Fisher Scientific), CD4 (GK1.5, Thermo Fisher Scientific), CD8a (53.6.7, Thermo Fisher Scientific), F4/80 (A3-1, Bio-Rad), MHC2 (M5/114.15.2, Thermo Fisher Scientific), CD11b (M1/70, Thermo Fisher Scientific), B220 (RA3-6B2, Thermo Fisher Scientific) CD11c (N418, Thermo Fisher Scientific), LY6C (HK1.4, Biolegend), LY6G (1A8, Biolegend), CD45 (30F11, Biolegend), Samples were acquired on a FACS Canto II (BD Biosciences) and analyzed with FlowJo software (FlowJo LLC).