Investigating the Cell Origin and Liver Metastasis Factors of Colorectal Cancer by Single-Cell Transcriptome Analysis

Abstract

Background

Colorectal cancer (CRC) is one of the deadliest causes of death by cancer worldwide. Liver metastasis (LM) is the main cause of death in patients with CRC. Therefore, identification of patients with the greatest risk of liver metastasis is critical for early treatment and reduces the mortality of patients with colorectal cancer liver metastases.

Methods

Initially, we characterized cell composition through single-cell transcriptome analysis. Subsequently, we employed copy number variation (CNV) and pseudotime analysis to delineate the cellular origins of LM and identify LM-related epithelial cells (LMECs). The LM-index was constructed using machine learning algorithms to forecast the relative abundance of LMECs, reflecting the risk of LM. Furthermore, we analyzed drug sensitivity and drug targeted gene expression in LMECs and patients with a high risk of LM. Finally, functional experiments were conducted to determine the biological roles of metastasis-related gene in vitro.

Results

Single-cell RNA sequencing analysis revealed different immune landscapes between primary CRC and LM tumor. LM originated from chromosomal variants with copy number loss of chr1 and chr6p and copy number gain of chr7 and chr20q. We identified the LMECs cluster and found LM-associated pathways such as Wnt/beta-catenin signaling and KRAS signaling. Subsequently, we identified ten metastasis-associated genes, including SOX4, and established the LM-index, which correlates with poorer prognosis, higher stage, and advanced age. Furthermore, we screened two drugs as potential candidates for treating LM, including Linsitinib_1510, Lapatinib_1558. Immunohistochemistry results demonstrated significantly elevated SOX4 expression in tumor samples compared to normal samples. Finally, in vitro experiments verified that silencing SOX4 significantly inhibited tumor cell migration and invasion.

Conclusion

This study reveals the possible cellular origin and driving factors of LM in CRC at the single cell level, and provides a reference for early detection of CRC patients with a high risk of LM.

Keywords:

• colorectal cancer
• liver metastasis
• single-cell sequencing
• SOX4
• prognostic

Ethical Approval and Consent to Participate

The study was approved by the ethics committee of Eastern Hepatobiliary Surgery Hospital, Naval Medical University.

The ethical considerations related to the dataset used in this study are described below. GSE178318: All patients who provided specimens signed an informed consent form and agreed to the specimens being used for scientific research. PRJNA748525: The written informed consent was received from each patient prior to participation. The study was approved by the Ethics Committee of Zhongshan Hospital and recorded by Ministry of Science and Technology of the People’s Republic of China. PMC9811165: This study was conducted in compliance with the Helsinki Declaration. All patients were enrolled according to a study protocol approved by the Stanford University School of Medicine Institutional Review Board. Written informed consent was obtained from all patients.

Acknowledgments

This study did not receive any specific funding.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors affirm that there are no potential conflicts of interest that could be perceived in terms of business or financial relationships in the conduct of this study.