MYO1F-KO neutrophil promoted tumor growth and inhibited anti-tumor response of CD8 ⁺ T cells . (A) WT and Myo1f^−/− C57BL/6 mice were subcutaneously injected with 1 × 10⁶ MC38 cells; tumor collection at day 21 (N = 5). (B) Tumor growth curve over time (N = 5). (C) Kaplan–Meier survival curves of MC38 tumor models (N = 10 in each group). (D) Gate strategy of tumor-infiltrating neutrophil flow sorting. (E) Spleen weight in B16F10 and MC38 tumor models of WT and Myo1f^−/− mice. (F) Immunofluorescence intensity of MYO1F analyzed by ImageJ; low and high were distinguished artificially according to the number of infiltrations. N (Low Neu infiltration) = 25, N (High Neu infiltration) = 20. (G) Gate strategy of intratumoral CD45.1⁺ neutrophil sorting from tumor. (H) Cell lysates of sorted tumor neutrophils were prepared and probed with antibody against MYO1F by immunoblotting. (I) Left: Gate strategy of the presence of total myeloid cells and neutrophils in BM at day 11 after irradiation without intervention. Right: Statistic of total myeloid cell and neutrophil counts in one tibia (N = 5). (J) Left: FACS analyses of intratumoral CD8⁺ (IFN-γ⁺, GZMB⁺, and Ki-67⁺) cells from tumor tissues. Right: Statistical analysis. (K) FACS analyses of intratumoral CD11b⁺Ly6G⁺ neutrophils from recipient tumor tissues. Right: Statistic of proportion and counts of neutrophils. (L) FACS analyses of intratumoral CD11b⁺Ly6G⁺ neutrophils from tumor tissues. (M) Kaplan–Meier survival curves of SX-682–treated B16F10 tumor models (N = 20 in each group). Data in A–E and G–L represent one experiment of three independent repeats; F represents one experiment of two independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (B, L, and M); log-rank (Mantel–Cox) (C); two-tailed unpaired Student’s t test (A, E, F, and I–K), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance. Neu, neutrophil. Source data are available for this figure: SourceData FS2.