Journal: Nature Communications

Article Title: The tumor microenvironment shows a hierarchy of cell-cell interactions dominated by fibroblasts

doi: 10.1038/s41467-023-41518-w

Figure Lengend Snippet: a Venn diagram of potential CAF-to-TAM ligands shared between breast cancer patient tumors and 4T1 tumors in mice based on the NicheNet ligand-receptor tool. b Heatmap representation of the expression of the 77 shared ligands from ( a ) in fibroblasts from the in vitro cell-circuit, based on bulk RNA-seq data from Fig. . c , d qPCR of Rarres2 and Cmklr1 from fibroblasts and macrophages. mono-cultured or co-cultured in control or cancer CM for 72 h. Data are combined from at least three independent experiments. P -value was calculated using two-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent ±SEM. c Fibroblasts mono-cultured in control or in cancer CM n = 5, fibroblasts co-cultured in control n = 5 or in cancer CM n = 4 biologically independent samples. d Macrophages mono-cultured in control n = 4 or in cancer CM n = 9, macrophages co-cultured in control or in cancer CM n = 7 biologically independent samples. e RARRES2 secretion levels in the media were assessed by ELISA from CAF n = 5 and 4T1 n = 4 biological biologically independent samples. P -value was calculated using two-sided students’ t test, Error bars represent ± SEM. f Transwell migration assay of macrophages in the presence of control medium n = 10, or 4T1 cancer CM with n = 9 or without n = 12 recombinant RARRES2 for 24 h, n indicates biologically independent samples. Luminescence values were normalized to 4T1-CM in log2. Data are combined from at least three independent experiments. P-value was calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Error bars represent ± SEM. g Violin plot of RARRES2 expression based on human scRNA-seq of normal fibroblasts n = 17 versus CAFs n = 32. P -value was calculated using two-sided students’ t test. h RARRES2 expression in different clusters from human scRNA-seq data, including CAF subclusters (defined using markers shown in Supplementary Fig. ). i Linear regression between RARRES2 expression in fibroblasts and TAM signature in macrophages. Each dot represents one patient, based on human scRNA-seq . P -value was calculated using F -test for linear regression. j RARRES2 gene expression in breast cancer patients stratified by grade, high grade = grade 3, n = 21; low-grade = grade 1 and 2, n = 10 patients. P -value was calculated using two-sided students’ t test. Source data are provided as a file.

Article Snippet: 4T1-GFP CM was added to the bottom chamber with or without 3 nM recombinant RARRES2 (R&D, 2325-CM-025).

Techniques: Expressing, In Vitro, RNA Sequencing Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Transwell Migration Assay, Recombinant