Abstract P5-06-05: Whole-genome bisulfite sequencing of single circulating tumor cells identifies cellular methylation heterogeneity in metastatic breast cancer
Authors: Luo Rui , Chong Weelic , Zhang Zhenchao , Abu-Khalaf Maysa , Silver Daniel , Fellin Frederick , Jaslow Rebecca , Lopez AnaMaria , Cescon Terrence , Ip Kevan , Myers Ronald , Wei Qiang , Li Bingshan , Wang Chun , Yang Hushan 
Published: March 2023

Abstract
Background: Although different patterns and changes in DNA methylation play an important role in cancer progression and tumor subtype differentiation, methylation remains relatively less understood in the context of tumor heterogeneity. Intra-tumor heterogeneity influences chemotherapy response, resistance development, and metastatic progression, and can be identified with liquid biopsy, a non-invasive approach to monitor cancer progression and provide predictive information. In this study, we sequenced circulating tumor cells (CTCs) to interrogate whole-genome methylation at single-cell level and single-base resolution. Methods: We enumerated CTCs in 7.5ml of whole blood samples from over 130 metastatic breast cancer patients using CellSearch, and selected blood samples with more than 20 CTCs to be further processed by DEPArray. After single-cell library construction using the Swift Accel-NGS Adaptase Module, whole-genome bisulfite sequencing (WGBS) was performed. The sequencing data were then aligned to the reference genome, and the methylation information was extracted by Bismark. We compared methylation profiles between CTC and WBC samples from each patient to identify differentially methylated regions (DMRs) using Methylkit. Multiple-comparison was conducted by SMART2 to identify DMRs within CTCs. Heatmap was plotted based on the regional methylation rates of DMRs. CpG and genic annotations were performed by annotatr. The pathway and function enrichment analyses (KEGG and GO) were conducted using clusterProfiler. Genomic region-based enrichment was conducted using LOLA. t-SNE clustering was used to investigate intra-patient heterogeneity. Results: A total of 376 cells from nine patients that passed our selection criteria were isolated for WGBS. The mean sequencing depth of all single cells was 0.8×, and an average mapping rate of 54% was achieved. We first compared CTCs and WBCs, and observed a global hypo-methylation pattern in CTCs (average methylation rate 68.8% in CTCs vs. 76.7% in WBCs). Moreover, approximately 2,000 highly differentially methylated regions were found in each patient, with hundreds of hypo- or hyper-methylated genes related to these DMRs. Hypo-methylated DMRs showed genomic region-based enrichment in breast-, prostate-, and fibroblast-related region sets. Then, we investigated methylation heterogeneity within CTCs, where t-SNE clustering identified four different subsets in one representative patient. About 1,500 hypo-methylated DMRs were found, differentiating those four subgroups. KEGG pathway analysis indicated enrichment in the Rap1 signaling pathway and focal adhesion. GO enrichment analysis highlighted the regulation of GTPase activity and membrane potential. Conclusions: Our study identified differentially methylated regions in CTCs of metastatic breast cancer patients, and demonstrated intra-patient heterogeneity based on cellular methylation information. Future studies are warranted to validate our findings and explore their biological mechanisms and clinical relevance.
Citation Format: Rui Luo, Weelic Chong, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Kevan Ip, Ronald Myers, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Whole-genome bisulfite sequencing of single circulating tumor cells identifies cellular methylation heterogeneity in metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-05.