INTRODUCTION AND OBJECTIVE:

Non-muscle invasive urothelial cancer is a heterogeneous disease with a variable history and differential response to chemotherapeutic and immunologically based therapy. Understanding of the molecular pathogenesis of NMIBC has expanded yet remains incomplete. Recent advances in single-cell RNA sequencing has yielded compelling insight into the biology of tumorigenesis and pathology of several malignancies and may provide greater understanding of this condition. The objective of this study was to demonstrate the feasibility of single cell analysis of fresh NMIBC specimens and initially compare the heterogeneity of low and high grade lesion metabolic pathway expression. METHODS:

We sequenced 65,000 cells per tumor from three low grade Ta and three high grade Ta human specimens. Single cell libraries were prepared using the 10X Genomics 3gene expression assay(v2) and high throughput sequencing was performed on a NovaSeq6000 to generate approximately 600 million reads-with close to 700 genes profiled per cell. 90% of reads mapped confidently to the human genome. Reads were processed using Cell Ranger (v2.2) and used Loupe Cell Browser (2.0) and Seurat(v2.2) to aggregate all data sets within and across the samples to perform differential gene expression analysis. RESULTS:

Using tSNE automated unbiased clustering of single cell gene expression we identified key subsets of carcinoma cells that were associated with markedly different functional states. Pathway enrichment increased several fold in the direction of the higher grade lesions. CONCLUSIONS:

Initial efforts at single cell RNA sequencing of NMIBC fresh tumors appears feasible with robust yields of cells and high quality control processing. Differences were noted in high grade and low grade lesions in the intensity of expression of several metabolic pathways including cellular oxidation and regulation of cell death with enrichment in the direction of higher grade lesions. Further analysis is warranted which may provide compelling insights into the heterogeneity and pathology of NMIBC.