Extended Data Fig. 7: Inflammatory and iron sequestering gene signature of LpIMB19-induced macrophages.
scRNA sequencing of flow cytometry-based sorted macrophages isolated from B16F10 melanoma-engrafted mice, 40 hrs after subcutaneous implantation of 5x106 B16F10 cells. a, Heat map showing relative expression of marker genes used for annotating three distinct subtypes of tumor-infiltrating macrophages. b, UMAP plots showing expression of inflammatory genes Nos2 (left) and Il1b (right) in macrophage subtype clusters. c, UMAP plots showing expression of anti- inflammatory genes Arg1 (left) and Mrc1 (right) in macrophage subtype clusters. d-j, Total n = 1062 cells for PBS and n = 1071 cells for RHP treatment group were analyzed. Cells were further categorized into subtypes based on marker genes expression as in a. For PBS control n = 399 regulatory TAM, n = 179 inflammatory macrophages, and n = 484 monocytic macrophages were analyzed; for RHP treatment n = 425 regulatory TAM, n = 357 inflammatory macrophages, and n = 289 monocytic macrophages were analyzed. d, Violin plots comparing expression of anti- inflammatory genes Arg1 (top) and Mrc1 (bottom) in macrophage subtypes treated with PBS or RHP. e-f, Violin plots comparing Tlr2 expression (e) and module score of Tlr2 signaling pathway (f) in macrophage subtypes. g-j, Violin plots showing module scores of signature genes in respective iron-related gene ontology biological processes (GOBP) within each condition for each macrophage subtype. k, LCN2 secretion by mouse peritoneal macrophages or bone marrow-derived dendritic cells, stimulated in vitro with LpIMB19 or RHP. n = 4 biological replicates, each representing co-culture setup with cells from one mouse. l, Western blot analysis of HO-1 in mouse peritoneal macrophages treated with LpIMB19 (MOI 1:30) and RHP (50 μg/ml) for 24, 48, 72 and 96 hrs. Results are pooled from two experiments (k) or are representative of two independent experiments (l). Statistical analyses were performed using two-tailed unpaired Student’s t-test (d-j) or one-way ANOVA with Tukey’s multiple comparison test (k).
