Fig. 5: Integrated spatial transcriptome and single-cell DNA sequencing analysis of a patient with PIK3CA mutation-positive ductal carcinoma in situ (DCIS) (Patient C).

a Visualization of the Visium results for Patient C. Hematoxylin and eosin staining (left). The number of spots in the tissue was 594. Spots were classified into two clusters via unhierarchical k-means clustering. The clusters almost completely overlapped with morphologically identified cancer cells (middle panel) and noncancer cells (stromal cells, right panel). b Coexpression network analysis of transcriptomes in DCIS spots. Weighted gene coexpression network analysis was applied to build the coexpression network and identify gene modules. The nodes and edges indicate genes and significant correlations between genes, respectively. In the top panel, the two nodes that appeared to have changed from normal cells are indicated by asterisks. Middle and lower panels present the subnetworks in modules (*1) and (*2), respectively. Node centrality, defined as the sum of strength, is represented by node size and color. c Pathway analysis was performed for each module using the Metascape tool. Each band represents one enriched term or pathway colored according to the −log 10 p value. Pathway enrichment analysis revealed that genes involved in p53 and estrogen signaling pathways were enriched in module 1, which shares the characteristics observed for the benign-appearing spots in Patients A and B, indicating that malignant transition had not occurred in this cancer. d Heatmap showing copy number variation at the single-cell level. The color scale for the copy number changes is shown at the right margin. The corresponding chromosomal locations are also shown. A total of 594 cells are represented. Clustering was performed as described in the “Methods”. Single-cell copy number variation analysis revealed two clusters, namely, clusters A and NC (noncancer). Cluster A features 1q+ and 16q− structural variants and represents DCIS cells. Because cluster NC does not have a structural variant, it was considered to represent noncancer cells from Patient C.