Fig. 2. SqD and UC regions of mixed histology tumors have distinct gene expression profiles.

a RNA sequencing analysis of 12 pairs of macrodissected UC and SqD regions from bladder tumors with mixed histology. Each region was classified using the TCGA molecular signature classification schema. All 12 SqD regions were basal/squamous subtype as were 8 of the 12 UC regions. Four tumors had discordant molecular subtypes. b Expression of BASE47 (left) and UPK/KRT (right) genes were evaluated by single sample GSEA analysis. Analysis was performed on 12 biologically independent UC-SqD pairs, Error bars represent standard error, p = 0.0005, two-sided paired Wilcoxon rank sum test. c Single-cell RNA-Seq analysis of a bladder carcinoma with extensive squamous differentiation revealed five clusters of tumor cells (clusters 1–5). d Trajectory analysis of these cell populations indicated a linear progression from cells resembling basal cells to those more similar to differentiated squamous cells. e Single-cell expression of select genes. Cluster 1 (61 cells) had an expression signature characteristic of the basal phenotype (high KRT5). Cluster 2 (64 cells) and Cluster 3 (152 cells) had expression signatures indicative of early squamous differentiation similar to that of suprabasal (spinous) cells (stratum spinosum), which are normally located immediately superficial to the basal cells of the epidermis (high DSC2, DSG2). Clusters 4 (231 cells) and 5 (220 cells) had a late squamous differentiation signature similar to that of granular squamous cells (stratum granulosum), which are normally located between the stratum spinosum and stratum corneum of the epidermis (high KRT13, KRT16).