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The primary contributing factors include the absence of reliable early detection methods, the anatomical intricacy of the biliary system, the inherently aggressive tumor biology, and the restricted effectiveness of systemic therapies. A profound understanding of molecular characteristics and clinically relevant emerging biomarkers is essential for advancing BTC treatment strategies. Recent developments in single-cell multi-omics technologies have enabled the analysis of genetic, transcriptomic, proteomic, and metabolomic data at the single-cell resolution, thereby uncovering the heterogeneity and complexity of tumor biology. These techniques provide critical insights into the diversity of immune cell populations within the tumor microenvironment (TME) and offer novel perspectives on tumor progression and potential therapeutic interventions. While single-cell technologies have significantly advanced the study of solid tumors, their application in BTC remains nascent, with a paucity of comprehensive reviews. This review systematically integrates single-cell genomics, transcriptomics, and epigenomics data to construct a cross-omics molecular atlas of BTC. It highlights the utility of single-cell multi-omics technologies in elucidating tumor heterogeneity, microenvironment remodeling, and clonal evolution in biliary tumors, while thoroughly analyzing their implications for clinical outcomes. Furthermore, this review explores personalized treatment strategies informed by single-cell technologies and underscores the significance of these technologies as indispensable tools for unraveling the complexity of BTC and fostering mechanism-based therapeutic innovation.<\/jats:p>","DOI":"10.1186\/s43556-025-00330-2","type":"journal-article","created":{"date-parts":[[2025,10,15]],"date-time":"2025-10-15T04:01:51Z","timestamp":1760500911000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Single-cell multi-omics in biliary tract cancers: decoding heterogeneity, microenvironment, and treatment strategies"],"prefix":"10.1186","volume":"6","author":[{"given":"Nannan","family":"Tang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiatong","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ao","family":"Gu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7054-448X","authenticated-orcid":false,"given":"Mengyao","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingbin","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,10,15]]},"reference":[{"issue":"1","key":"330_CR1","doi-asserted-by":"publisher","DOI":"10.1186\/s40779-024-00535-6","volume":"11","author":"X-B Zhang","year":"2024","unstructured":"Zhang X-B, Fan Y-B, Jing R, Getu MA, Chen W-Y, Zhang W, et al. 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