The Interaction between Macrophages and Triple‐negative Breast Cancer Cells Induces ROS‐Mediated Interleukin 1α Expression to Enhance Tumorigenesis and Metastasis

Abstract Triple‐negative breast cancer (TNBC) has higher mortality than non‐TNBC because of its stronger metastatic capacity. Increasing studies reported that TNBC tumors had more macrophage infiltration than non‐TNBC tumors, which promoted the metastasis of TNBC cells. However, how TNBC cells become more malignant after interacting with macrophages is less reported. In this study, it is observed that when TNBC cells are co‐cultured with macrophages, they display higher viability and stronger metastatic ability than non‐TNBC cells. Mechanistic studies reveal that TNBC cells acquired these abilities via interactions with macrophages in three phases. First, within 12 h of co‐culture with macrophages, some TNBC cells have significantly elevated levels of reactive oxygen species (ROS), which upregulate interleukin 1α (IL1α) expression in ERK1/2‐c‐Jun‐ and NF‐κB‐dependent manners at 24−48 h. Second, the secreted IL1α bound to IL1R1 activates the ERK1/2‐ZEB1‐VIM pathway which increases metastasis. Third, IL1α/IL1R1 facilitates its own synthesis and induces the expression of IL1β and IL8 at 72−96 h through the MKK4‐JNK‐c‐Jun and NF‐κB signaling pathways. Moreover, a higher level of IL1α is positively correlated with more macrophage infiltration and shorter overall survival in breast cancer patients. Thus, reducing ROS elevation or downregulating IL1α expression can serve as new strategies to decrease metastasis of TNBC.

culture was 67 and the number of cancer cells at initial was 2000.tdT: red fluorescent protein; GFP and clover: green fluorescent protein; C3: apoptotic sensor cells emit green fluorescence in live cells and emit blue fluorescence in apoptotic cells.

Figure S2 .
Figure S2.Macrophages injected alone into the nude mice failed to form any lung colonies and TNBC cells induced macrophage polarization to M2 type.(A) qPCR results showing the mRNA levels of M1 macrophage markers TNF, NOS2, CD86 and M2 macrophage markers of CD163, CD204 and CD206 in Raw264.7 cells before and after 96 h of co-culturing with 231-GFP or MCF-7 cells.The results represent the means ± SD from three independent experiments or form six mice.(B) Schematic diagram of the tail vein injection with mono-or co-cultured cells.(C) Representative lung images from the mice injected with mono-or cocultured Raw264.7-tdTmacrophages alone or Raw264.7-tdTco-cultured with 231-GFP cells through the tail vein for 28 days.(n = 6 mice per group).Scale bar: 1 mm.(D) Percentage of the colony area formed by macrophages to the total left lung area of each treatment group.Significant differences were determined by one-way ANOVA.**P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure S3 .
Figure S3.Identification of the driver genes that were upregulated in the co-cultured TNBC cells.(A) Volcano diagram of the differentially expressed genes.Red dots indicate upregulated genes, and blue dots indicate downregulated genes with the cut-off criteria of fold change > 1 and P < 0.05.(B) GO enrichment analysis of the upregulated signaling pathways.(C and D) qPCR validation of the top 10 upregulated secreted genes in the 231-GFP or MDA-MB-468 cells after being co-cultured with Raw264.7-tdTmacrophages.(E) qPCR results of the knockdown efficiency of the target genes in the 231-GFP cells with shRNAs.The results represent the means ± SD from three independent experiments.Significant differences were determined by Student's t-test.*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Figure S4 .
Figure S4.Determining the importance of the candidate genes in enhancing the viability of 231-GFP cells and migration ability of Raw264.7 macrophages.(A) Representative fluorescence images of the tumor spheres formed by the monocultured 231-GFP cells with or without the target gene knockdown.Scale bar, 200 m.(B and C) Schematic diagram (C) and representative images (B) of the Transwell analysis of the Raw264.7 macrophages with the

Figure S5 .
Figure S5.Body weights of NOD/SCID mice implanted with orthotopic tumors.Body weight of the NOD/SCID mice inoculated with mono-or co-cultured THP1-tdT macrophages and 231-GFP cells with or without the knockdown of IL1, IL1 and IL8.(n = 6 NOD/SCID mice per group).

Figure S6 .
Figure S6.ROS elevation activated the ERK1/2-c-Jun and NF-B signaling pathways to upregulate the expression of IL1, IL1 and IL8 in TNBC cells.(A) Western blotting of the protein levels of p-MKK4, JNK and p-JNK in the mono-or co-cultured 231-GFP cells.(B) Western blotting of c-Jun, p-c-Jun, IL1, IL1 and IL8 in the 231-GFP cells with or without the treatment of the c-Jun inhibitor SR11303 (5 M) during 96 h of the co-culture treatment

Figure S7 .
Figure S7.Knockdown of IL1R1 reduced the viability and migration of the 231-GFP cells.(A and B) qPCR and Western blots showing the mRNA and protein levels of ILR1 in the 231-GFP cells transfected with IL1R1 shRNA.(C) (left) Representative fluorescence images of the tumor spheres formed by the 231-GFP cells with or without the IL1R1 knockdown when cocultured with Raw264.7-tdTcells.Scale bar, 200 m.(right) Quantification of the total green fluorescence intensity indicated the viability of the 231-GFP cells on day 7 compared to day 1. (D) Representative images and the quantified results of the Transwell migration assay of the 231-GFP cells with or without the IL1R1 knockdown.Scale bar, 200 m.(E) Body weight of the NOD/SCID mice inoculated with co-cultured THP1-tdT macrophages plus 231-GFP cells

Figure S8 .
Figure S8.IL1 activated three signaling pathways mediated by ERK1/2, MKK4 and NF-B during the co-culture of TNBC cells with macrophages.(A) Western blots showing the total or phosphorylated protein levels of IL1, IL1, IL8, ERK1/2, ZEB1, VIM, MKK4, JNK, c-Jun, IB and NF-B in the MDA-MB-468 cells before and after 96 h of the co-culture treatment with Raw264.7-tdT or THP1-tdT macrophages.(B) Western blots showing the total

Figure S9 .
Figure S9.Overexpression of IL1 upregulated IL1 and IL8 and promoted the survival, migration, invasion and lung metastatic ability of 231-GFP cells.(A) qPCR results showing the mRNA levels of IL1, IL1 and IL8 in the 231-GFP cells with or without the overexpression of IL1.(B) Representative fluorescence images of the tumor spheres formed by the 231-GFP cells with or without the overexpression of IL1 when co-cultured with