Extended Data Fig. 2: Characterization of CTC-associated WBCs.

a, Bar plot showing the expression levels of WBC marker CD45 in patient samples, including CTC-associated WBCs (red), free-floating peripheral WBCs (blue) and CTCs alone (green). b, Principal component analysis (PCA) of CTC-associated WBCs of patients and five reference WBC populations (n = 50). c, Bar plot showing the expression levels of CD45 in mouse samples, including CTC-associated WBCs (red) and CTCs alone (green). d, PCA of CTC-associated WBCs from all mouse models and five reference WBC populations (n = 47). e, Reference component analysis clustering of CTC-associated WBCs (red) and reference WBCs from mouse models, displaying projection scores of cells (columns, n = 47) on the immune reference panel (rows). f, Representative immunofluorescence images of mouse immune cells stained for CD45, Ly-6G and CD11b. Mouse lymphocytes (CD45⁺Ly-6G^–CD11b^–), mouse monocytes (CD45⁺Ly-6G^–CD11b⁺), and mouse granulocytes (CD45⁺Ly-6G⁺CD11b^low) are shown (top). Representative immunofluorescence images of mouse lymphocytes and macrophages (peritoneum-derived) stained for F4/80 are shown (bottom). Mouse macrophages display a F4/80⁺ phenotype, whereas lymphocytes display a F4/80^– phenotype (n = 3 for all). g, Representative images of human and mouse T cells, B cells, NK cells, monocytes and granulocytes stained with the Wright–Giemsa protocol to highlight nuclear morphology (left). Wright–Giemsa staining of the human CTC-derived cell line BR16 is also shown (right) (n = 3 for all). h, Representative immunofluorescence images of CTC–neutrophil clusters and CTC–monocyte clusters isolated from mouse models and stained for Ly-6G (gold) and CD11b (blue). CTCs stably express GFP (green). n = 8 for NSG-CDX-BR16-GFP, NSG-LM2-GFP and NSG-4T1-GFP, n = 7 for BALB/c-4T1-GFP. i, Representative images of CTC–neutrophil clusters stained with the Wright–Giemsa protocol to highlight nuclear morphology. n = 8 for NSG-CDX-BR16-GFP, NSG-LM2-GFP and NSG-4T1-GFP. j, Bar graph showing the mean number of CTC–neutrophil clusters and CTC–monocyte clusters in mouse models. Error bars, s.e.m.; n = 8 for NSG-CDX-BR16-GFP, NSG-LM2-GFP, NSG-4T1-GFP and n = 7 for BALB/c-4T1-GFP. k, Heat maps showing the projection scores of mouse-derived (left) and patient-derived (right) CTC-associated neutrophils (columns) on pro-tumoral (N2) neutrophil markers (rows).

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