Abstract 857: Identify ROR1 expression in healthy human tissues using single-cell RNA sequencing

Abstract

Abstract Introduction: The Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) is a receptor for Wnt5a and is considered a tumor-specific antigen. Researchers are actively exploring the development of antibodies and adoptive cell therapies targeting ROR1, while the potential "on-target, off-tumor" toxicity is still a major concern. Previous studies have provided inconsistent results regarding the expression of ROR1 in normal tissues, highlighting the complexity of its expression profile. Therefore, we are conducting this study to address this challenge through the detection of ROR1 mRNA expression via single-cell RNA sequencing (scRNA-seq) and the evaluation of ROR1 protein levels by immunohistochemistry (IHC). Methods: We retrieved the Gene Expression Omnibus (GEO) database and downloaded the scRNA-seq data of each organ. We used the Seurat package to normalize data, perform dimensionality reduction and conduct clustering. The differentially expressed genes of each cell cluster were identified, and the cell clusters were then manually annotated. We further conducted the IHC on the tissue microarray (TMA), and evaluated the ROR1 immunostaining. Results: We analyzed the scRNA-seq data of 908,941 cells from 14 types of healthy human organs, and 132,837 (14.61%) of cells expressed ROR1. Cell clusters in which the organ's ROR1 positive rate was >1% included the human Heart (Overall 25.99%, Ventricular cardiomyocyte 70%, Atrial cardiomyocyte 50%, Mesothelial 37% and Adipocytes 27%), Pancreas (Overall 9.36%, Beta cell 12.4%, Alpha cell 9.1%, Duct cell 7.3%, Acinar cell 7.2% and Delta cell 7.2%), Colon and small intestine (Overall 6.09%, Paneth 14.9%, Intermediate absorptive enterocyte 14.4%, Transit amplifying 13.8% and Enteroendocrine cell 12.5%), Lung (Overall 3.86%, Alveolar type I 23%, Ciliated cells 5.8% and Alveolar type II 5.4%), Bladder (Overall 2.73%, Fibroblasts 6.8% and Myofibroblast 6.4%), Esophagus (Overall 1.93%, Epithelial suprabasal 5% and Epithelial basal 4.4%) and Stomach (Overall 1.31%, Gland mucous cell 3% and Pit mucous cell 1.9%). The ROR1 expression was found relatively low (<1%) on Adipose, Kidney, Ileum, Liver, Spleen, Bone marrow and PBMC. For the IHC, ninety normal tissues were prepared and used. Consistent with the scRNA-seq results, we found relatively high ROR1 protein on the Heart, Lung, Stomach, Duodenum and Colon and low ROR1 protein on Liver and Ileum. However, ROR1 protein was not detected on Bladder, Pancreas or Esophagus during the IHC process. Conclusion: Our findings suggest that some healthy human tissues may be at risk of "on-target, off-tumor" toxicity when utilizing ROR1-targeted therapies, including Heart, Colon and small intestine, Lung and Stomach. Close monitoring of possible toxicities related to the high ROR1 expressing organs should be considered in future clinical trials. Citation Format: Mingyang Feng, Sirui Tan, Yue Chen, Qiu Li, Yongsheng Wang. Identify ROR1 expression in healthy human tissues using single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 857.