Fig. 5: CCL26 is required for OXTR^High CAFs function.

a Cytokines secreted from OXTR^Low and OXTR^High CAFs sorted from three individual CAF cell lines (CAF1-3), displayed as a venn diagram, with the numbers of overlapping cytokines indicated (left image). The numbers of shared cytokines elevated in all OXTR^High CAFs are indicated (n = 23). b The scan map of Human Cytokine array (middle) (n = 3 independent samples for each group). Table (right) displays seven cytokines/chemokines which were significantly differently upregulated (fold change >2) in the media of OXTR^High CAFs. c Relative expression (RT-PCR) of seven cytokines/chemokines after OXTR knockdown in OXTR^High CAFs. P = multiple t test. n = 4 for each group. d, e Representative images of DiI-labeled conditional CAFs (red) co-cultured with CMFDA-labeled HN6 (green) to form heterotypic spheroids for organoid invasion studies. Cultured cells were supplemented with recombinant human CXCL6, CCL26, CCL18, IL-1β, CCL7, CCL5, or LIGHT or stable knockdown of each of seven cytokines/chemokines. Scale bar: 100 μm. f, g Graphical representation of maximal distance of spheroid invasion from the spheroid border (Lmax) in (d, e) and quantification using ImageJ software, n = 5/group. P = two-tailed t test. h, i Representative H&E images of 3D organotypic cultures of CAFs/tumor cells under the conditions indicated and used to estimate the ability of tumor invasion, displayed graphically. Scale bar: 100 μm. j, k The invasive index was calculated using ImageJ software. n = 3/group, P = two-tailed t test. l Graphical presentation of the invasion index of HN6 OSCC cells with CCR3 knockdown co-cultured in the 3D Organotypic system used in (h) with OXTR^Low CAFs and CCL26, n = 4/group. P = two-tailed t test. m IHC analysis for CCR3 expression in OSCC tumor microenvironment. Scale bars, 200 μm (n = 10). Scale bar: 100 μm. n Graphical representation of the correlation between EMT pathways from a GSEA analysis of CCR3^High versus CCR3^Low HNSCC patients, n = 522 (TCGA, Firehose Legacy). o The correlation between CCR3 expression and recurrence-free survival in HNSCC patients (n = 124) using Kaplan–Meier plotter (http://kmplot.com/analysis/). Results are shown as mean and standard deviation (SD) of three independent experiments. Source data are provided as a Source data file.