Fig 1: Leading edge heterogeneity revealed by ST. A) H&E staining of tissue sections (left) and mapped with unbiased clustering of ST spots in 4 tumor samples (right). Each region is surrounded by dotted lines in different color. Scale bar = 500 µm. B) t-SNE plot of 11 137 ST spots from four primary breast cancer ST data. Each cluster is shown in different color. C) MIA map of overlap between all scRNA-seq-identified epithelial cell clusters and ST-identified spot clusters. Each element in the matrix is computed for all pairs of epithelial cell clusters and spot clusters using the same 728 background genes. Red indicates enrichment (significantly high overlap); blue indicates depletion (significantly low overlap). D) All ST spots of BMS score and the tumor leading edge ST spots of BMS and EMT score in T3 and T4 tissue sections. The intensity represents score of each ST spot. Scale bar = 500 µm. E) Violin spots of BMS score of the tumor inner region and the tumor leading edge in T3, T4, and four samples together. F) Unbiased clustering of ST spots mapped to T3 and T4 tissue sections, respectively (left, middle). BMS score feature plots from T3 and T4 with data generated using the Visium ST platform. The intensity represents max expression of each gene. Scale bar = 500 µm. (right). G) Violin plots of BMS score by ST spots subpopulation in ST data. H) Bar plots of GSVA analysis comparing between leading edge-associated ST plots and non-leading edge-associated ST plots in four primary breast tumor samples. All p-values were determined using an unpaired two-sided Wilcoxon rank-sum test. ns, p = 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. BMS, breast metastatic signature. COX6C, cytochrome C Oxidase Subunit 6C. DHRS2, dehydrogenase/Reductase 2. EMT, epithelial–mesenchymal transition. GSVA, Gene set variation analysis. H&E, Hematoxylin and eosin. MIA, multimodal intersection analysis. ST spatial transcriptomics
Fig 2: Clinical significance of OXPHOS in early disseminated breast cancer. A–C) Dot plots showing significantly higher level of OXPHOS and lower level of glycolysis in N status positive (N1-3) breast cancer patients than those with N status negative (N0). Patients sequence data were from FUSCC (A), TCGA (B), and METABRIC (C). All p-values were determined using an unpaired two-sided Wilcoxon rank-sum test. D) UMAP plot of 6350 cancer epithelial cells with tissue source. Each tissue source is shown in different color. E) Bubble plot of enriched KEGG pathways of cancer epithelial cell from lymph nodes compared to those from primary tumors. The intensity represents adjusted p-value of each KEGG pathway. Dot size shows gene count for each KEGG pathway. Wilcoxon signed-rank test was used to assess the difference. F) Kaplan–Meier curve illustrating higher COX6C and DHRS2 accompanied by poor OS in basal-like breast cancer patients from TCGA dataset. G) Kaplan–Meier curve illustrating higher COX6C accompanied by poor OS and DMFS in HER2-positive breast cancer while higher DHRS2 upregulation was associated with poor OS and DMFS in basal-like breast cancer. Log-rank test was used to assess the difference. ns, p = 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; **** p < 0.0001. DMFS, distant metastasis-free survival. GOBP, Gene Ontology Biological Process. KEGG, Kyoto Encyclopedia of Genes and Genomes. OS, overall survival. OXPHOS, oxidative phosphorylation. UMAP, Uniform Manifold Approximation and Projection.
Fig 3: Downregulation of COX6C and DHRS2 inhibited breast cancer cells proliferation, migration, and EMT. A) Line plots showing significantly lower cell proliferation rates in T-47D and MDA-MB-231 cells after knocking down COX6C and DHRS2. B) Bar plots showing downregulation of COX6C and DHRS2 significantly inhibited cell migration ability of T-47D and MDA-MB-231 cells in trans-well assay (right). Representative images randomly selected from T-47D and MDA-MB-231 cells are shown (left). Scale bars = 1 mm. C) IHC images of COX6C and DHRS2 expressions in primary tumor with or without lymph node metastasis. Three independent experiments were performed and generated similar results. Scale bar = 100 µm. D) Western blot images showing the EMT signaling pathway was inactivated in shCOX6C and shDHRS2 group, compared with control of MDA-MB-231 and 4T1 cells. E) Scatter plots with a significant positive spearman correlation for the expression of COX6C and those of DHRS2. F) Violin spots of EMT score of the high expression and the low expression of COX6C and DHRS2 in epithelial cells. All p-values in (A) and (B) were determined using an unpaired two-sided Student's t-test. All p-values in (F) were determined using an unpaired two-sided Wilcoxon rank-sum test. Data presented as the mean ± SD. of n = 3. ns, p = 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. EMT, epithelial–mesenchymal transition. shCOX6C, COX6C knockdown. shDHRS2, DHRS2 knockdown.
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