Fig 1: APOC1+APOE+ macrophages mediate the establishment of immune microenvironment of lymph node metastasis in ESCC. a) Representative images of multiplexed IF staining of tumor and metastatic lymph node samples using Panel-1 and Panel-2. Scale bar, 50 µm. b) Cell density of CD3+CD4+FoxP3+ Treg cells, exhaustive CD8+PD1+ T cells, CD68+CD163+ macrophages, CD20+ B cells in both tumor nest, and stroma between primary tumor and metastatic lymph node (n = 50). c) Representative images of IHC staining of APOE and APOC1 in metastatic lymph node and primary tumor. Scale bar, 100 µm. d) Cell percentage of APOE+APOC1+ macrophages in in tumor and metastatic lymph node (n = 58). e) Kaplan–Meier curve of overall survival of ESCC patients stratified by the abundance of APOE and APOC1 in an independent cohort. f) Spatial characterization of APOE+APOC1+ macrophages in tumor and metastatic lymph node by multiplexed IF staining. Scale bar, 50 µm. g) Cell density of APOE+APOC1+ macrophage in both tumor nest and stroma between primary tumor and metastatic lymph node. h) Correlation between CD163+ macrophages and APOE+APOC1+ macrophages. i) Representative images of IHC staining of DSG2, DSG2, SDC4, COL6A1, COL6A2, CXCL9, CXCL10, CXCR3, ITGA11, Siglec1, and SPN in metastatic lymph-node and primary tumor (n = 20). Scale bar, 100 µm. j) Coexpression of Siglec1 (red) with SPN (green) in metastatic lymph node were shown by double IF staining. Cell nuclei were stained with DAPI (blue). The white arrows indicate the location of Siglec1 and SPN positive cells. Scale bar, 50 µm. Data are presented as means ± SD (standard deviation). TN: tumor nest, PT: primary tumor, LNM: metastatic lymph node, M?: macrophages.