Fig. 4 | Nature Communications

Fig. 4

From: Macrophages orchestrate breast cancer early dissemination and metastasis

Fig. 4

Early disseminated cancer cells contribute to metastasis formation. Macrophages were depleted from pre-malignant MMTV-HER2 mice by ASF98 treatment starting at week 18. Treatment was stopped when mice developed palpable tumors (1–3 mm average). a Mice were left until tumors reached 1 cm in diameter and then sacrificed. b Time from beginning of treatment at age wk18 until development of palpable tumors as mean±SEM (9 mice each, 23–38 wks old). c Time from formation of palpable tumors until tumors were overt (26-43 wks old) as mean±SEM (control N = 9, anti-CSF1R N = 6). Macrophages in sections of overt tumors (at least three animals per group) were identified by staining against F4/80 at the end of the experiment (d, e; bar: 100 μm; zoom factor in insets 5x) and quantified as the number of macrophages relative to tumor area (f; statistical analysis: Mann–Whitney test). Vascularization of overt tumors was analyzed by staining against endomucin, an endothelial cell marker (g, h; bar: 100 μm; zoom factor in insets 4x) and quantified as endomucin+ area/tumor in at least three animals combined (i; mean±SEM; statistical analysis: Mann–Whitney test). Solitary DCCs in lung sections and metastases defined as cell clusters bigger than three cells were quantified in lung sections stained against HER2 (jl, bar: 25 μm). For solitary cell analysis, the average of DCCs or metastases per 100 fields was counted; each dot represents one lung section. For metastasis analysis, the total number of metastases per lung sections was quantified and plotted (j). Number of mice N = 6 (control) and N = 4 (αCSF1R) animals combined; statistical analysis: Mann–Whitney test