Fig. 3: Genetic deletion of selected chemosensors on tumor-conditioned macrophages.

a–e, FACS analysis of macrophages in the absence (LGP) or presence (OLFR644^−/−) of Olfr644 deletion and Vmn2r29 deletion (Vmn2r29^−/−). Macrophages were exposed to Pten^−/−Trp53^−/− conditioned media. Events were gated on F4/80⁺CD11b⁺ cells. Bar graphs show ratios between conditions: mean fluorescence intensity of CD206 (a); percentages of CD206^brightMHCII⁻ (b) and CD206⁻MHCII⁺ (c) cells gated on F4/80⁺CD11b⁺ cells (LGP n = 4, Vmn2r29^−/− n = 4, Olfr644^−/− n = 4); and percentages of ARG1⁺ (d) and CD39⁺ (e) cells gated on F4/80⁺CD11b⁺ cells (LGP n = 4, Vmn2r29^−/− n = 5, Olfr644^−/− n = 3). f, Venn diagrams showing common and specific genes among the differentially expressed genes calculated for the three conditions: LGP + Pten^−/−Trp53^−/− conditioned media versus LGP; OLFR644 + Pten^−/−Trp53^−/− conditioned media versus LGP + Pten^−/−Trp53^−/− conditioned media; VMN2R29 + Pten^−/−Trp53^−/− conditioned media versus LGP + Pten^−/−Trp53^−/− conditioned media. g, Graphs showing change in expression of selected genes among differentially expressed genes from the three comparisons (red, upregulated; blue, downregulated). h, Proliferation of CD8⁺ T cells exposed to supernatant from Untr. and CM-tr. macrophages: the bar graph shows the number of divisions (LGP Untr. n = 5, LGP CM-tr. n = 4, Vmn2r29^−/− Untr. or Cm-tr. n = 6, Olfr644^−/− Untr. or Cm-tr. n = 6). i, Scratch assay: graph and curves showing the distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages (LGP n = 6, VMN2R29 n = 6, OLFR644 n = 6). j,k, Flow cytometry analysis to assess the impact of chemosensor gene silencing on macrophage phenotypes. Olrf644^−/− and Vmn2r29^−/− macrophages were compared to control macrophages (LGP) after exposure to conditioned media from ovarian ID8 cancer cells (LGP n = 3, NT n = 3, Vmn2r29^−/− n = 4, Olfr644^−/− n = 3) (j) or breast 4T1 cancer cells (LGP n = 3, NT n = 3, Vmn2r29^−/− n = 3, Olfr644^−/− n = 3) (k). Events were gated on F4/80⁺CD11b⁺ cells. Statistical analyses were performed using two-tailed unpaired Student’s t-test. Values are presented as the mean ± s.e.m. All replicates represent biological replicates.