Extended Data Fig. 8: Transcription factors, mIHC and in vitro validation of neutrophil clusters.

a, UMAP plots showing regulon activities of five representative transcription factors for specific neutrophil clusters. Binding motifs of these transcription factors are shown on the top. b, Normalized ATAC-seq sequencing tracks of selected transcription factor loci in matched PBN, ALN, and TAN isolated from the same patient. ATAC peaks detected by MACS3 are denoted with the grey box above the gene body and highlighted with light red shading. c, Workflow of co-culture experiments of PBNs with or without cell line (liver cancer cell line HepG2, HCCLM3, and MHCC97H, control cell line HEK293T). d, Survival curve of PBN in culture condition (n = 3, n denotes biologically independent samples). Data are presented as mean values ± SEM. e, Expression of TAN-related signatures in PBNs co-cultured with or without different cell lines for 0 h, 18 h, 24 h, and 30 h. f, Expression of gene signatures of different neutrophil subsets in PBNs co-cultured with or without different cell lines. g, White arrows mark CCL4⁺CD66b⁺ neutrophils, with one cell highlighted by the four enlarged panels on the right. Middle panels show another representative CCL4⁺ CD66b⁺ neutrophil while right panels show a representative CCL4⁻ CD66b⁺ neutrophil. Scale bars are 20 μm and 2 μm. h, Expression of selected genes in PBNs co-cultured with or without different cell lines for 0 h, 18 h, 24 h, and 30 h. i, Chord diagrams showing interactions between neutrophils and other cell types mediated by CCL3-CCR1 and CCL4-CCR5. Line width is proportional to interaction intensity, coloured by cell types with receptors. j, Crystal violet staining of migrated monocytes co-cultured with matched TAN or non-TAN. Scale bar, 100 μm. k, FACS analysis showing the PD-L1 expression of PBNs co-cultured with or without different cell lines for 24 h. Two-way ANOVA test is used for e and h.

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