CX3CL1 is a mediator of a cold TME in human cancers. (A) Representative immunofluorescence staining of CX3CL1 in an autochthonous DMBA/TPA skin carcinoma, showing distinct regions of high and low CX3CL1. Blue scale bar = 500 µm, white scale bar = 100 µm. Representation of three tumors. (B) Quantification of F4/80⁺ macrophages per mm² in CX3CL1 high and low regions of autochthonous DMBA/TPA skin carcinomas (n = 3 tumors, 15–28 tiles counted for each region). (C) Representative immunofluorescence staining of F4/80 in regions of low (top) and high (bottom) CX3CL1. Scale bar = 100 µm. Representation of three tumors. (D–G) Analysis of immune cells and CX3CL1 expression using a previously published dataset of 364 human tumors from 12 cancer types. Computational clustering of tumors using flow cytometry data and transcriptomic data enabled identification of 12 dominant patterns of immune composition across various cancers, referred to as immune archetypes (Combes et al., 2022). RNA-seq data were generated separately for all viable cells and for sorted mononuclear phagocytes. Prior to RNA-seq, cells were sorted by flow cytometry from untreated, freshly resected human tumors. (D) T cell score of each tumor, based on RNA-seq of all viable cells, grouped on the x axis by immune archetype (n = 5–24). (E) Heatmap of average CX3CL1 RNA expression, based on RNA-seq of all viable cells, for each immune archetype (n = 5–28). (F) Ratio of macrophage score and monocyte score, based on RNA-seq of mononuclear phagocytes, for tumors in the five cold immune archetypes (n = 5–24). (G) CD206^Hi macrophage score, based on RNA-seq of mononuclear phagocytes, for tumors in the five immune cold archetypes (n = 5–24). (H) Ratio of macrophages to monocytes (left) and CD206^Hi macrophages to non-CD206^Hi macrophages (right) detected in mouse CIT6-YFP tumors overexpressing Cx3cl1 or controls (n = 11). Data are representation of one experiment. Statistical significance was determined by two-way ANOVA with Tukey’s correction posttest (B), Mann–Whitney test with the Bonferroni correction (F and G), or Student’s t test (H). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.