Fig 2: Colorectal cancer produces an ectopic source of epithelial hepcidin that portends decreased patient survival.(A) Representative Prussian blue iron staining and (B) Quantification in human colorectal tumor and normal adjacent tissue (Normal N=13 and Tumor N=15 biologically independent samples, p=0.001). (C) Representative staining for ferroportin in colorectal tumor and normal adjacent tissue from a mouse model of sporadic colorectal cancer (N=4 biologically independent normal and tumor samples). (D) Kaplan-Meier survival analysis generated from 530 human CRC biopsies, stratified on high vs. low hepcidin transcript from the median. (E and F) qPCR analysis for hepcidin (Hamp) expression in normal and tumor tissue in sporadic (E) and inflammation-associated (F) mouse models of colorectal cancer (N= 5 and N=12 independent animals, respectively; p=0.046 and p=0.0316, respectively). (G) Experimental design for tumor epithelium vs. stroma enrichment. (H) qPCR analysis for Hamp in epithelium vs stroma compartments of normal and tumor tissue (N=3 independent animals with sporadic colorectal cancer). (I) Experimental design for fluorescence-activated cell sorting gated on live cells and Epcam intensity in tumor stroma (Epcam-) and epithelia (Epcam+) from a mouse model of sporadic colorectal cancer. (J) qPCR analysis for Hamp in epithelium vs stromal compartments of normal and tumor tissue (N=3 biologically independent animals with sporadic colorectal cancer). Data represent the mean ± SEM. Significance was determined by 2-tailed, unpaired (B) or paired (E and F) t test. Survival analysis significance was assessed by Cox proportional hazards regression (D). *p < .05, ****p <.0001.

Fig 3: Cat dander extract induces epithelial plasticity and activation of NFκB/RELA. C57BL/6J mice were treated with daily challenges of CDE or PBS i.n. over 4 days. A Changes in mesenchymal markers. Relative fold changes of indicated transcripts by Q-RT-PCR of total lung RNA; *p < 0.05, compared to Control, n = 6–8/group, t-test. B IFM. Sections were stained with anti-SNAI1 Ab (red) and DAPI (blue) for nuclear staining. Representative images shown at × 20 with selected enhanced image of the epithelium. C Relative fold changes in SNAI1 FI for each treatment group (n = 6–8 animals/group). D Single cell flow cytometry using a 4-day CDE exposure model. Representative gating of cell selection, removal of debris, doublets and Live-CD45− cells. [Left] a representation of SMA+ and CD326+ control cells vs [Right] SMA+ and CD326+ CDE-exposed cells. E % SMA+ and CD326+ cells in the 4-day CDE exposure model, n = 5. F Sections stained with anti-RELA Ab (red) and DAPI (blue) for nuclear staining. Representative images shown at × 20 with selected enhanced image of the epithelium. G Relative fold changes in RELA FI for each treatment group (n = 6–8 animals/group)

Fig 4: CD14 is upregulated in CD45+CD11b+ leukocytes in the embryonic stroma following LPS stimulation. (A) Histogram showing CD326 expression in primary adult or embryonic intestinal stromal cells, with intestinal epithelial cells as a positive control. Data representative of n = 3 independent cultures generated from 3 adult mice or 3 litters of 6 embryos each. (B,C) Flow cytometric analysis of CD45 and CD11b expression in adult or embryonic intestinal stromal cells. The percentage of live CD45+CD11b+ cells were compared. n = 3 biological replicates. t-test ***p < 0.001. (D) Histogram depicting CD14 expression in CD45-CD11b− or CD45+CD11b+ populations in adult or embryonic intestinal stromal cells treated with LPS or vehicle control. (E) CD14 mean fluorescence intensity (MFI) in CD45+CD11b+ gated adult or embryonic intestinal stromal cells treated with LPS or vehicle. n = 3 biological replicates. One way ANOVA with Tukey’s multiple test. ****p < 0.0001.

Fig 5: BORG expression correlates with stem-like phenotypes in TNBC cells. A, D2.OR cells co-stained with fluorescently-labeled antibodies against α6 integrin (CD49f) and EpCAM were sorted by flow cytometry to isolate “differentiated” cell (e.g., α6 integrinLow) or “stem-like” cell (e.g., α6 integrinHighEpCAMHigh populations. B, Sorted D2.OR cells were plated at varying concentrations in nonadherent cell culture plates, and mammosphere forming frequency was calculated on the basis of binary ability to form a sphere in each well. C, BORG expression was quantified via qRT-PCR using RNA derived from parental D2.OR cells, D2.OR cells enriched for a “differentiated” population (e.g., α6 integrinLow), and mammospheres propagated from D2.OR cells. (±SE; *, P < 0.05). D, MDA-MB-231 cells co-stained with fluorescently-labeled antibodies against CD44 and CD24 were sorted by flow cytometry into “stem-like” (CD44HighCD24Low) and “non-stem” (CD24High) populations. E, BORG expression was quantified via qRT-PCR in human mammary epithelial cells (HMEC) or MDA-MB-231 parental cells, “stem-like” cells (CD44hi/CD24lo), and “non-stem” cells (CD24hi; ±SE; *, P < 0.05). F, D2.OR cells engineered to ectopically express BORG are enriched for a transcriptional signature that is found to be overexpressed in mammary stem cells. G, Aldh1a3 transcript expression is significantly higher in D2.OR cells overexpressing BORG, as compared with D2.OR cells expressing an empty vector (±SE; *, P < 0.05). H, ALDH activity was quantified via the ALDEFLUOR assay in D2.OR cells expressing BORG or an empty vector. DEAB+ signifies addition of ALDH inhibitor to establish background fluorescence control. DEAB− signifies active ALDH catalysis of fluorescent substrate. Percent cells with detectable ALDH activity annotated. I, Correlation of BORG and Nanog expression raw count values in a collection of 28 various human breast cancer cell lines (r = 0.97). J, Single-cell RNA-seq of 11 primary TNBC samples from two publicly available datasets (PRJNA305054; PRJNA485429) was analyzed for coexpression analysis. The five genes with the strongest coexpression pattern with BORG (expression > 0.4) in individual TNBC tumor cells are shown as a heatmap, with Nanog ranked number 2 (P < 0.0002). K, Nanog expression was quantified in BORG-expressing or parental D2.OR cells via qRT-PCR (±SE; *, P < 0.05).