Adam Karami

"Single cell transcriptomic analysis reveals expansion of unique epithelial cell populations and during esophageal carcinogenesis"

Introduction: Esophageal squamous cell carcinoma (ESCC) is among the deadliest of human malignancies. Despite advances in understanding the genetic molecular features of ESCC, poor prognosis and therapy resistance remain significant clinical challenges. As defining cell types involved in ESCC progression may unveil novel therapeutic targets, we utilized single cell RNA-sequencing to define alterations in esophageal epithelium during 4-Nitroquoline 1-oxide (4NQO)-mediated ESCC carcinogenesis. 

Methods: Peeled esophageal epithelium was collected from C57/B6 mice before 4NQO treatment (Normal; n = 12), after 16 weeks of 4NQO (Precancer; n = 6) or after 21 weeks of 4NQO followed by a 5 week wash out period (Cancer; n = 4). RNA was extracted and libraries were prepared with the InDrop protocol. UMI-tools identified cell barcodes and UMIs from raw reads. UMI-extracted reads were aligned by STAR. Alignments were assigned to genes with FeatureCounts. UMI-tools retrieved counts for each gene in each cell. Analysis of count matrices was done with R package Seurat. Cells with mitochondria gene expression >20% were filtered out and datasets were batch corrected. UMAP was used to find principal components and reduce dimensionality. Monocle calculated reversed graph embedding for pseudotime projection. 

Results: Visualization with UMAP embeddings distinguished epithelial cells from mice with normal esophageal epithelium from those exposed to 4NQO. 8 epithelial subsets were identified based upon transcriptional profiles with expression of respective basal and superficial markers Krt5 and Krtdap guiding identification of 5 basal, 1 suprabasal, and 2 superficial clusters Although absent in normal esophageal epithelium, basal clusters 3, 4 and 5 as well as superficial cluster 2 expanded in esophageal epithelium from 4NQO-treated mice. By contrast, the following clusters were diminished in 4NQO-treated mice: basal 2, suprabasal and superficial 1. Pseudotime analysis across the dataset revealed an initial branch point at which normal basal cells marked by high Krt5 and low Krtdap transitioned toward either differentiation or a cancer lineage. the latter enriched for basal clusters 3, 4 and 5. Cells moving toward the cancer lineage then further branched toward proliferation or a differentiated lineage terminating in superficial cluster 2. 

Conclusions: Esophageal epithelium undergoes remarkable alterations in its cellular landscape during 4NQO-mediated carcinogenesis. Two basal cell clusters and one superficial cluster emerged in precancerous expanded during ESCC carcinogenesis. Notably, these basal and superficial clusters are respectively enriched at the predicted terminal proliferative and differentiated cell fates in ESCC and may represent targets for therapy in this deadly malignancy.