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Recent advances in single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) have enabled high-resolution profiling of OS tumors, revealing diverse malignant, immune, and stromal cell populations. These studies have identified proliferative, inflammatory, and angiogenic tumor states, immunosuppressive myeloid subsets, exhausted T cells, and complex cell\u2013cell communication networks that contribute to tumor progression and immune evasion. However, several challenges constrain the broader application of single-cell approaches in OS. The mineralized structure of bone tissue complicates dissociation into viable single cells, and the rarity of OS limits access to fresh specimens. Most existing datasets are based on small, heterogeneous cohorts and are generated using diverse protocols, which complicate data integration and comparison. Emerging methods are beginning to overcome these barriers. These include snRNA-seq for frozen and archival tissue, improved dissociation protocols for mineralized tumors, and integration with spatial transcriptomics to retain spatial context. Moving forward, combining single-cell transcriptomics with complementary modalities, such as immune repertoire analysis, chromatin accessibility profiling, and spatial proteo-genomics, combined with functional validation, will provide deeper insights into immune dynamics, regulatory mechanisms, and the cellular architecture of the OS TME. This review summarizes the current landscape of single-cell transcriptomics in OS and highlights methodological challenges in single-cell studies in OS tumors, recent biological insights, and their implication for immunotherapies.<\/jats:p>","DOI":"10.1007\/s10555-026-10336-z","type":"journal-article","created":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T01:44:18Z","timestamp":1778118258000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["New biological insights into osteosarcoma\u2014lessons from single cell sequencing studies"],"prefix":"10.1007","volume":"45","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-6942-6866","authenticated-orcid":false,"given":"Julia I.","family":"Zehenter","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4954-6488","authenticated-orcid":false,"given":"Leo","family":"Kager","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9061-0504","authenticated-orcid":false,"given":"Sebastian K.","family":"Eder","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1439-5301","authenticated-orcid":false,"given":"Sabine","family":"Taschner-Mandl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5549-7737","authenticated-orcid":false,"given":"Sne\u017eana","family":"Hini\u0107","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2026,5,7]]},"reference":[{"issue":"1","key":"10336_CR1","doi-asserted-by":"publisher","DOI":"10.1038\/s41572-022-00409-y","volume":"8","author":"HC Beird","year":"2022","unstructured":"Beird, H. 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