Fig 1: C3a receptor antagonism results in tumor growth delay of Pan02 syngeneic mouse model in C57BL/6 mice and is mediated by increased NK cell infiltration in the TME. A, After Pan02 tumor cells implanted subcutaneously in C57BL/6 mice reached a volume of approximately 100 mm3, treatment was started with either daily intraperitoneal injection of vehicle (4% DMSO in PBS; control group, n = 6) or with daily intraperitoneal injection of the small-molecule C3aR antagonist SB290157 (SB290157 group, 5 mg/kg, n = 6) for 22 days and tumor size was monitored during and after treatment until endpoint (tumor ulceration or volume >1,000 mm3). The mean time to reaching 300 mm3 in the control group was 11.2 days compared with 23 days in the SB902157 treatment group (****, P < 0.0001). B, Flow cytometry analysis of control versus C3aR antagonist treated Pan02 tumor-bearing mice. Mice were randomized in two treatment groups (control vs. C3aR antagonist, n = 5 per group) and were treated daily for six treatments and euthanized the day after the last treatment. C3aR antagonism results in an increased NK cells (NK1.1+; as a percentage of live cells) in the TME (**, P = 0.0032). Representative gating of NK cells shown (*, Proportion of NK cells as a percentage of live cells). C, Representative IF staining of control and C3aR antagonist–treated mice (n = 3 per treatment group), showing an increase of NK cells (NK1.1-FITC) in the TME of C3aR antagonist-treated mice compared with vehicle-treated mice. D, Flow cytometry analysis of control versus C3aR antagonist–treated Pan02 tumor-bearing mice. Except a decrease in macrophages (Gr1-CD11b+F4/80+) in the C3aR antagonist–treated tumors (**, P = 0.0055), no statistically significant (ns) difference in B cells (B220+), CD4+ T cells, CD8+ T cells, and MDSCs (Gr1+CD11b+) is noted between the two treatment groups. E, Left: NK and CD8+ T cells depletion experiment scheme. After randomization into the treatment arms, NK cells or CD8+ T cells depletion was achieved by the administration of 100 µg of antibody (anti-NK1.1, or anti-CD8) intraperitoneal on days 0, 4, 8, and 12. Mouse IgG1 was used as isotype control. Mice undergoing NK and CD8+ T cells depletion and receiving IgG1 isotype control antibody were treated with daily intraperitoneal injection of SB290157 (5 mg/kg) from day 0 to day 13. Mice in the control arm were treated with daily vehicle (4% DMSO in PBS). Right: Depletion of NK cells abrogates the effect of the C3aR antagonist, resulting in tumor growth rate similar to the control group, whereas with CD8+ T cells depletion, C3aR antagonist retains its effect on tumor growth delay. Statistical significance determined using t test.