Fig. 2: P. anaerobius drives MDSC migration by activating the integrin α2β1–NF-κB–CXCL1 pathway in CRC cells.
a, Representative images of MDSCs that migrated to the lower chamber and quantification of MDSC migration towards CRC-cell-conditioned medium pre-incubated with vehicle or P. anaerobius (n = 3 biologically independent samples). Scale bars, 100 μm. b, Cytokine array analysis (36 cytokines) of conditioned medium from Caco-2 cells treated with P. anaerobius or vehicle (left). Fold change in cytokine levels after P. anaerobius treatment (right). Red box, significantly changed cytokines; NC, negative control. c, Levels of CXCL1 secreted from Caco-2 and MC38 cells treated with vehicle or P. anaerobius (left). Levels of serum CXCL1 and mRNA expression of CXCL1 in the colon of Apcmin/+ mice (right). d, Integrin α2β1 knockdown in Caco-2 cells abrogated P. anaerobius-induced CXCL1 secretion (left) and migration of MDSCs (right). e, RGDS peptide treatment of MC38 cells abrogated P. anaerobius-induced CXCL1 secretion (left) and migration of MDSCs (right). f, Treatment with JSH-23 abolished P. anaerobius-induced CXCL1 secretion (left) and MDSCs migration (right). c–f, n = 3 biologically independent samples. g–j, AOM-induced CRC mice were treated with RGDS. g, Tumour load of the mice; n = 8 (PBS), 11 (PA), 7 (PBS + RGDS) and 10 (PA + RGDS) mice. h, Serum levels of CXCL1; n = 5 (PBS and PA) and 4 (PBS + RGDS and PA + RGDS). i, Percentage of MDSCs in the colonic lamina propria of the mice (left). Immunofluorescence images (middle) and quantification of CD11b+Gr-1+ cells in mouse tumours (right). Scale bars, 25 μm. j, Mean fluorescence intensity (MFI) of IFN-γ+CD8+ and IFN-γ+CD4+ T cells in colonic lamina propria. i,j, n = 7 (PBS and PBS + RGDS) and 9 (PA and PA + RGDS) mice. k–m, MC38 allograft model mice were treated with RGDS peptide and anti-PD1. k, Tumour growth curves (left) and tumour weight (right); n = 7 (PBS + anti-PD1) and 8 (all other groups) mice. l, CXCL1 levels in tumour tissue lysates; n = 5 (PBS + IgG, PBS + anti-PD1 and PBS + anti-PD1 + RGDS), 8 (PA + IgG), 7 (PA + anti-PD1) and 3 (PA + anti-PD1 + RGDS) mice. m, Percentage of MDSCs (left) and IFN-γ+CD8+ T cells (right) in tumours; n = 7 (PBS + anti-PD1) and 8 (all other groups) mice. a,c–m, Data are the mean ± s.e.m. P values were calculated using a one-way ANOVA, followed by Tukey’s post-hoc test (a,d–j); an unpaired two-tailed Student’s t-test (c); a one-way ANOVA, followed by Fisher’s least significant difference test (k–m) or a two-way ANOVA, followed by Bonferroni’s post-hoc test (k).
