Fig 1: CXCL8 is higher expressed and secreted in CAAs compared with NAs.A Heatmap showing the transcriptomic expression levels of the secretory proteins in 10 NA and 10 CAA tissues. B Volcano plots depict DEGs of secretory proteins. C, D GO and KEGG enrichment scatter plot for upregulated transcriptome genes for the secretome in CAAs compared with NAs. E, F GSVA and GSEA pathway enrichment analysis of upregulated transcriptome genes for the secretome. G Heatmap of the top 20 differentially expressed transcriptome genes in the secretome. H ELISAs detected 11 secretory proteins in NA and CAA supernatants. I Two overlapping sets with Veen diagrams of upregulated secretory proteins between RNA-seq and ELISA. J, K IHC and IF staining of CXCL8 in 10 NA and 10 CAA tissues. L IF staining of NAs and CAAs. M, N CXCL8 gene and protein expression in MDA-MB-231 cells after coculture without or with NAs or CAAs for 7 days. O GSEA of the “INTERLEUKIN-8_PRODUCTION” gene set in the CAAs versus NAs. P Overall survival analysis of independent breast cancer cohort GSE20685 for the prognostic potential of CXCL8. CAA cancer-associated adipocyte, CXCL8 C-X-C motif chemokine ligand 8, DEGs differentially expressed genes, ELISA enzyme-linked immunosorbent assay, GSEA gene set enrichment analysis, GSVA gene set variation analysis, IF immunofluorescence, IHC immunohistochemistry, NA normal adipocyte. Data are presented as the means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: Working model.In TNBC, CAA is positively associated with an immunosuppressive microenvironment. CAA is highly expressed and secretes CXCL8, which promotes tumor growth and lung metastasis and regulates EMT through the PI3K/AKT pathway. CXCL8 modulated the immunosuppressive microenvironment by upregulating CD274 expression and suppressing T-cell infiltration. Blockade of the CAA-derived CXCL8 pathway can sensitize the immunotherapy response of PD-1, thus inhibiting TNBC progression. CAA cancer-associated adipocyte, NA normal adipocyte, PD-1 programmed cell death protein 1, CD274 programmed death-ligand 1.
Fig 3: CXCL8 aggravates TNBC proliferation, migration, invasion, and EMT via the PI3K/AKT pathway.A Viability of MDA-MB-231 cells treated with different concentrations of CXCL8. B, C Representative images of wound healing and Transwell assays of MDA-MB-231 cells after treatment with CXCL8 (10 ng/ml) or CXCL8 (10 ng/ml) + CXCL8 antibody (10 μg/ml) for 7 days. D Viability of 4T1 cells treated with different concentrations of MIP2. E, F Representative images of wound healing and transwell assays of 4T1 cells after treatment with MIP2 (20 ng/ml) or CXCL8 (20 ng/ml) + MIP2 antibody (10 μg/ml) for 7 days. G, H Western blot and IF analysis of the EMT-related markers AKT and p-AKT in MDA-MB-231 cells after treatment with CXCL8 (10 ng/ml) or CXCL8 (10 ng/ml) + CXCL8 antibody (10 μg/ml) or CXCL8 (10 ng/ml) + GSK2141795 (10 μmol/L) for 7 days. I, J Western blot and IF analysis of the EMT-related markers AKT and p-AKT in MDA-MB-231 cells after treatment with MIP2 (20 ng/ml) or MIP2 (20 ng/ml) + MIP2 antibody (20 μg/ml) or MIP2 (10 ng/ml) + GSK2141795 (10 μmol/L) for 7 days. CXCL8 C-X-C motif chemokine ligand 8, DEGs differentially expressed genes, ELISA enzyme-linked immunosorbent assay, EMT epithelial–mesenchymal transition, GSEA gene set enrichment analysis, GSVA gene set variation analysis, IF immunofluorescence, IHC immunohistochemistry, MIP2 macrophage inflammatory protein-2, TNBC triple-negative breast cancer. Data are presented as the means ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
from Cell Signaling Technology for Human Interleukin-8 (hIL-8) Recombinant Protein