Fig 2: TLR3 is a major mediator of IFN-β production and autocrine signaling. A BT-549 cells were infected with lentiviruses encoding OSM or shRNAs targeting either TLR3 or GFP. Following selection, cells were assessed by qRT-PCR and B Western blot analyses for the efficiency of TLR3 knockdown. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via Welch’s t-tests where *p < 0.05. C qRT-PCR and D Western blot analyses of BT-549-shTLR3, BT-549-shGFP, and BT-549-OSM cells assessing IFN-β1, ISGs, IFNARs, and ISGF3. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via Welch’s t-tests where *p < 0.05. E E0771 cells were infected with lentiviruses encoding OSM or shRNAs targeting either Tlr3 or GFP. Following selection, cells were assessed by qRT-PCR for efficiency of Tlr3 knockdown. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via Welch’s t-tests where **p < 0.01. F qRT-PCR and G Western blot analyses of E0771-shTlr3, E0771-shGFP, and E0771-OSM cells assessing Ifn-β1, ISGs, IFNARs, and ISGF3. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via Welch’s t-tests where *p < 0.05 and **p < 0.01. H Transwell migration of BT-549-shTLR3, BT-549-shGFP, and BT-549-OSM cells was assessed for 72 h via Incucyte imager. Data is graphed as mean ± SD, n = 6. Statistical significance was determined via two-way ANOVA with multiple comparisons where ****p < 0.0001. I Transwell migration of E0771-shTlr3 and E0771-shGFP cells was assessed for 84 h via Incucyte imager. Data is graphed as mean ± SD, n = 6. Statistical significance was determined via two-way ANOVA with multiple comparisons where **p < 0.01

Fig 3: A novel interferon gene signature correlates with patient prognosis. A RNA-sequencing data was analyzed to identify common differentially expressed genes between BT-549-OSM, BT-549-TGF-β1, BT-549-SNAI1, and BT-549-ZEB1 cells relative to BT-549-Vec cells. Only genes with p values < 0.05, as determined by the DESeq2 package in R, were included. B Differentially regulated genes in BT-549-OSM, BT-549-TGF-β1, BT-549-SNAI1, and BT-549-ZEB1 cells compared to BT-549-Vec cells are organized into 3 categories: consistently upregulated in each group, consistently downregulated in each group, or inconsistently regulated between groups compared to BT-549-Vec cells. C Kaplan–Meier plot of patients with TNBC, D HER2 + breast cancer, E gastric cancer, and F colon cancer that express high or low levels of the 50 repressed genes from A using the median as the cutoff value for expression

Fig 4: OSM inhibits IFN-β production and autocrine signaling in TNBC. A BT-549 cells were infected with lentiviruses encoding OSM or control (Vec). Following selection, cells were assessed via Western blot analysis for OSM and STAT3 phosphorylation. B RNA-sequencing was performed on BT-549-OSM and BT-549-Vec cells and pairwise comparisons of the sequencing data were used to assess interferon-family genes. C Western blot and D qRT-PCR analyses of BT-549-OSM and BT-549-Vec cells assessing IFN-β1, ISGs, IFNARs, and ISGF3. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via Welch’s t-tests where *p < 0.05 and **p < 0.01. E GSEA of RNA-sequencing data of BT-549-OSM and BT-549-Vec cells was performed using an experimentally-derived IFN-β metagene signature. A false-discovery rate (FDR) correction was applied to the statistical significance. F E0771 cells were infected with lentiviruses encoding OSM or control (Vec). Following selection, cells were assessed via Western blot analysis for STAT3 phosphorylation. G qRT-PCR and H Western blot analyses of E0771-OSM and E0771-Vec cells assessing Ifn-β1, ISGs, IFNARs, and ISGF3. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via t tests where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig 5: EMT-inducing factors inhibit IFN-β production and autocrine signaling. A BT-549 cells were infected with lentiviruses encoding OSM, TGF-β1, SNAI1, ZEB1, or a control vector (Vec). Following selection, cells were assessed via western blot for the indicated targets. B qRT-PCR and C Western blot analyses of cells from A assessing IFN-β1, ISGs, IFNARs, and ISGF3. D–F GSEA of RNA-sequencing data from the cells in A was performed using an experimentally-derived IFN-β metagene signature; D BT-549-TGF-β1, E BT-549-SNAI1, F and BT-549-ZEB1 cells were compared to BT-549-Vec cells. A false-discovery rate (FDR) correction was applied to the statistical significance. G qRT-PCR and H Western blot analysis of cells from A assessing TLR3 expression. Data represents mean fold changes ± SEM, n = 4. Statistical significance was determined via Welch’s t-tests where **p < 0.01