Trastuzumab efficacy on HER2⁺xenografts also relies on neutrophils, and FcγRs and Syk are required in neutrophils for mAb-induced antitumor activity. (A-C) Indicated mice (n ≥ 4) were injected subcutaneously with 5 × 10⁶ BT474-luc2 cells in matrigel at day 0, intravenously with 100 μg of Trastuzumab or isotype Ctrl weekly starting day 1, and total photon flux was acquired (photons per second). Mice were also injected (A) on days −1, 1, 3, 5, and 7 with anti-Gr1 mAbs. (B) Data are compiled from 2 identical experiments. (Nota bene: Anti-Gr1 mAb-treated nude mice and littermates, Gfi1^−/− nude, and Gfi1^+/− nude littermates were kept under sulfamethoxypyridazin plus trimethoprim.) (D) FcRγ^−/− mice (n ≥ 4) were injected with B16-luc2 cells and mAb TA99 as in Figure 1, daily with 2 × 10⁶ neutrophils purified from indicated mice (▲: neutrophil injection). (E) Ex vivo cytotoxicity of human neutrophils (PMNs) on opsonized-BT474-luc2 cells at a 50:1 effector:target ratio. (Note: BT474 cells express HER2 but not CD20; thus anti-CD20 Rituximab [Rituxi.] represents a negative control. Triton lysis of BT474-luc2 is used as a positive control. Mean of triplicates is represented.) (F) H&E staining or anti-Gr1 immunolabeling, or (G) DAPI staining and anti-Ly6G immunolabeling of sections of 7-day-old B16-luc2 tumors 24 hours after mAb TA99 injection. (F) Original magnification, ×10 (scale bar = 100 μm). (H) Mice (n ≥ 4) were injected with B16-luc2 cells, mAb TA99, and analyzed as in Figure 1. (A-E,H) Data are represented as mean ± SEM (not significant: P > .05; *P < .05; ***P < .001) and are representative from at least 2 independent experiments. Ctrl, control; DAPI, 4′6 diamidino-2-phenylindole; H&E, hematoxylin and eosin; PMN, neutrophils; Trastu, Trastuzumab.