Single-cell analyses reveal metastasis mechanism and microenvironment remodeling of lymph node in intrahepatic cholangiocarcinoma

Background & Aims: Lymph node metastasis (LNM) is a major determinant of recurrence and prognosis in intrahepatic cholangiocarcinoma (iCCA). LNM disrupts T cell-mediated cytotoxicity, promotes tumor-specific immune tolerance, and facilitates distant metastasis. Despite its importance, extensive research on LMN in iCCA is lacking. This study aimed to systematically explore the heterogeneity of the LNM-associated microenvironment in iCCA by integrating single-cell and multi-omics analyses, identifying metastasis-associated cell subgroups, and validating these findings through multiple cohorts. Methods: We analyzed single-cell transcriptomics data from primary tumors, cancer-adjacent liver tissues, and tumor-draining lymph nodes of four patients with iCCA who underwent radical surgery. Additionally, we collected 81 tumor and matched lymph node tissue sections from patients with iCCA. We performed single-cell RNA sequencing and multiplex immunohistochemistry, followed by differential gene expression analysis, functional enrichment analysis, single-cell copy number variation assessment, and pseudotime analysis. Results: Our analysis revealed the complex heterogeneity of the iCCA LNM-associated microenvironment. We found a significant increase in stromal and mature immune cells in the metastatic lymph nodes. T cells constitute the predominant component, with diverse functional subtypes. We identified CD36+ macrophages and SAA1+ tumor cells as key players in the metastatic process. The SAA1-CD36 receptor-ligand pair may be crucial in forming the LNM-associated microenvironment. Conclusions: We identified several metastasis-associated cell subgroups. These findings provide new insights into the mechanisms underlying LNM in iCCA and lay the groundwork for the development of novel therapeutic strategies. Our study highlights the importance of single-cell technologies in understanding tumor microenvironment complexity and offers valuable resources for future research. (c) 2024 The Author(s). Published by Elsevier B.V. on behalf of European Association for the Study of the Liver (EASL). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).