Additional file 1 of Iron diminishes immunosuppressive macrophages and enhances anti-PD-1 immunotherapy in breast cancer models
Year: 2026
Type: article
Abstract: Supplementary Material 1: Figure S1. The association between iron metabolism and immunosuppression in TAMs of human breast cancer. A, Bubble plot showing the expression levels of signature genes associated with immunosuppression. B, Correlation between enrichment scores for iron uptake and transport, and immunosuppression signatures across all cells colored by cell type based on the study by Wu et al, 2020. Each point represents a single cell. Cell type classification and detailed patient information can be found in the source study. C, Bubble plot showing the expression levels of major iron and heme metabolic genes in the indicated cell types. D, Bubble plot showing the expression of marker genes for identifying the subtypes of myeloid cells. Dot color indicates expression level and size indicates the proportion of cells expressing each gene. E and F, Violin plots showing enrichment scores for immunosuppression signature (E), and iron uptake and transport pathway signature (F) among the identified cell types from the indicated clinical subtypes of breast cancer based on the study by Wu et al, 2021. The red dashed line shows the mean of the enrichment scores. G, Correlations between enrichment scores for iron uptake and transport and immunosuppression signatures across all cells (colored by cell type) from the indicated clinical subtypes of breast cancer based on the study by Wu et al, 2021. Figure S2. Analysis of single-cell RNA-Seq data from E0771 breast cancer model. A, Clustering and UMAP plots showing all retained cells merged (left) and retained cells from each E0771 tumor/sample (right 3 panels) for analysis after scRNA-seq. B, Bubble plot showing the expression levels of signature genes associated with immunosuppression. C, Correlations between enrichment scores for iron uptake and transport and immunosuppression signatures across all cells colored by cluster (left) and colored by cell type (right). Each point represents a single cell. Figure S3. In vitro iron treatment had no significant effect on viability of TNBC cells with clinically relevant doses. A and B, Effect of iron treatment (with doses from 0.5 to 50 mM) on viability of TNBC cells HS578T (A) and MDA-MB-468 (B). Representative cell growth plots where each point represents the mean ± SD (n = 5-6 per condition). Experiments were repeated at three independent times with similar results. **, P< 0.01 versus other treatment at day 3, by two-tailed unpaired Student’s t-test. Figure S4. Single-cell RNA-Seq profiling to characterize tumor infiltrated immune cells after iron supplementation in E0771 model. A, Gating strategy for flow sorting of viable tumor infiltrating CD45+ cells. B, Clustering and UMAP plots of scRNA-seq data showing cells isolated from E0771 tumors with indicated treatment. Figure S5. Characterization of T and NK cells after iron treatment in E0771 and PyMT tumors. A, UMAP plot and clusters of unbiased sub-clustering of T and NK cells in E0771 tumors. B, Bubble plot showing the expression levels of marker genes for the subtype annotation of T and NK cells as clustered in A. C, Bubble plot showing the expression levels of cytotoxic signature genes in each subclusters as clustered in A. D, Representative images and quantification of CD8 and granzyme B immunofluorescence staining in MMTV-PyMT tumors with the indicated treatments. Arrows indicate CD8 and granzyme B double-positive cells. Scale bar, 50 mm. Error bars represent SEM. P-value determined by Student’s t-test. Figure S6. Iron supplementation diminishes immunosuppressive phenotype of TAMs. A, Bubble plot showing the expression levels of major iron metabolic genes in TAMs of E0771 tumors with indicated treatment. Dot color indicates expression level and size indicates the proportion of cells expressing each gene. B, Violin plots showing enrichment scores of immunosuppression gene signature among TAM, monocyte and neutrophil clusters with indicated treatment. ns, not significant. *, P < 0.01 and **, P < 1 x 10-5 vs PD-1 by two-tailed Student’s t-test. The red dashed line shows the mean of enrichment score. C-E, Analysis of PD-L1 levels in TAMs and tumor cells of E0771 model after the indicated treatments by flow cytometry. C, Gating strategy. D and E, representative histograms and quantification of PD-L1 expression by mean fluorescence intensity (MFI) in TAMs (D) and tumor cells (E). Error bars represent SD. P-value determined by one-way ANOVA with Tukey’s test.
Source: Figshare
Language: English
Topic: Immune cells in cancer
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7138906556