Distinct cellular trajectories diverge between cancerous and non-cancerous pathologies in the murine

Adam Karami, MS(1); Mohammed Faujul Kabir, PhD(1); Alena Klochkova, MS(1); Anbin Mu(1); Yinfei Tan, PhD(2); Andres Klein-Szanto, MD, PhD(2); Kelly A. Whelan, PhD(1) (1)Fels Cancer Institute for Personalized Medicine, Temple University, Philadelphia, PA (2)Department of Pathology and Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA

Epidemiological data indicate that eosinophilic esophagitis (EoE) patients fail to develop esophageal malignancy. Our previous studies pairing MC903/ovalbumin (OVA)-mediated EoE and 4-nitroquinoline 1-oxide (4NQO)-mediated esophageal squamous cell carcinoma (ESCC) in mice indicate that exposure to EoE inflammation inhibits esophageal carcinogenesis. As both EoE and ESCC feature epithelial remodeling, we hypothesized that differential alterations in epithelium may contribute to EoE protective effects against malignancy. Here, we use single-cell RNA-sequencing to analyze cellular heterogeneity in mice with EoE and ESCC. C57B6 mice were treated with MC903/OVA for 32 days to induce EoE or 4NQO for 24 weeks to induce ESCC. InDrop libraries were prepared from esophageal epithelial RNA. UMI-tools identified likely cell barcodes, which were STAR-aligned assigned to genes. The R program Seurat was used to find principal components and reduce dimensionality, visualized by UMAP plots. Monocle3 was used to calculate reversed graph embedding for pseudotime. Unsupervised classification resolved 28 distinct epithelial cell clusters. 13 basal, 8 suprabasal, and 7 superficial clusters were identified using the expression gradients of basal marker Krt5 and superficial marker Krtdap. 5 populations unique to EoE and 4 populations unique to ESCC were identified. Pseudotime further revealed cell fate trajectories between conditions. In mice with ESCC, a basal-to-differentiated cell trajectory as found in untreated esophagi was apparent; however, 3 basal and 1 suprabasal subpopulations diverged. EoE-specific populations were enriched with limited cells marked as basal or superficial, suggesting impaired squamous differentiation. These data demonstrate that EoE and ESCC have distinct effects on cellular heterogeneity in esophageal epithelium, with aberrant basal and suprabasal populations emerging in the context of cancer and aberrant suprabasal populations emerging in the context of EoE inflammation. Future studies will explore cellular pathways that drive aberrant cell fates in these conditions as well as their functional role in the pathogenesis of EoE and ESCC.