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Unveiling the role of the extracellular matrix in the osteosarcoma tumor microenvironment through integrated transcriptomics and experimental validation

Cancer Gene Therapy (2025)Cite this article

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Abstract

Osteosarcoma (OS), the most common malignant bone tumor, is characterized by heterogeneous tumor cells and abundant microenvironmental components. The extracellular matrix (ECM)—a complex and dynamic network surrounding tumor cells—plays a pivotal role in OS malignancy (e.g., cell proliferation, metastasis), making insights into ECM involvement critical for advancing OS prognosis. This study conducted bioinformatic analyses on bulk RNA-sequencing and single-cell RNA sequencing data from public databases, initially identifying collagen type V alpha 2 (COL5A2) as a key gene in OS progression. It further validated biological functions and underlying mechanisms of COL5A2 via in vitro experiments, and constructed and validated prognostic models based on ECM signature cell clusters. Results identified osteoblastic cells (OCs) and endothelial cells (ECs) as core cellular components of OS. COL5A2 was highly expressed in OCs, and high COL5A2 expression correlated with significantly reduced overall survival in OS patients. Western blot, CCK-8, and colony formation assays demonstrated that COL5A2 promoted OS cell proliferation by activating the focal adhesion pathway and inducing phosphorylation of the FAK/Paxillin/Akt signaling axis. The prognostic model highlighted the C0 OCs cluster as clinically significant. CellChat analysis uncovered significant activation of the IGFBP pathway in both C0 OCs and C1 ECs, and identified the IGFBP3-TMEM219 axis as the key ligand-receptor pair mediating their crosstalk. This study establishes COL5A2 and the C0 OCs cluster as pivotal ECM-related signatures in OS, confirming COL5A2 drives OS proliferation through focal adhesion signaling and IGFBP3-TMEM219-mediated crosstalk—both representing promising therapeutic targets. Further investigation into ECM components is warranted to refine OS treatment strategies and improve clinical outcomes.

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Fig. 1: Overview of the single-cell landscape in OS tissues and normal tissues.
Fig. 2: Construction and validation of ECM signature gene RS in OS tissues.
Fig. 3: Expression patterns and validation of ECM signature genes in OS.
Fig. 4: Functional validation of COL5A2 in MG63 cells.
Fig. 5: Functional validation of COL5A2 in U2OS cells.
Fig. 6: Identification and heterogeneity of ECM-related ECs clusters.
Fig. 7: Identification and heterogeneity of ECM-related OCs.
Fig. 8: Construction and validation of prognostic models for ECM signature cell clusters.

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Data availability

All the datasets analyzed in this study are publicly available as described in the Materials and methods in the manuscript.

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Acknowledgements

This work was financially supported by National Natural Science Foundation of China (grant number: 82403886, 82002842), and the National Defense Science and Technology Fund for Distinguished Young Scholars (grant number: 2022-JCJQ-ZQ-018), and Innovation Science Fund of the Fourth Medical Center of PLA General Hospital (grant number: 2025-4ZX-MS-03).

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Author notes

  1. These authors contributed equally: Yuyang Liu, Yuchen Han, Zixuan Guo.

Authors and Affiliations

  1. Senior Department of Orthopedics, Chinese PLA General Hospital, Beijing, China

    Yuyang Liu, Yuchen Han, Zixuan Guo, Yinglong Zhang, Meng Xu & Jianxiong Li

  2. Department of Neurosurgery, 920th Hospital of Joint Logistics Support Force, Kunming, Yunnan, China

    Yuyang Liu

  3. Kunming Medical University, Kunming, Yunnan, China

    Yuyang Liu & Xiuyuan Xu

  4. Medical School of Chinese PLA, Beijing, China

    Yuchen Han, Zixuan Guo, Yinglong Zhang, Meng Xu & Jianxiong Li

  5. Department of Endocrinology, 920th Hospital of Joint Logistics Support Force, Kunming, Yunnan, China

    Xiuyuan Xu

  6. Department of Internal medicine, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Wenting Qi

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Contributions

Yuyang Liu and Yuchen Han: Data analysis, experiments, writing review and editing. Zixuan Guo: Investigation and methodology. Yinglong Zhang and Xiuyuan Xu: Investigation and writing review. Jianxiong Li, Meng Xu and Wenting Qi: Designed the study, funding acquisition, project administration, supervision, data analysis and interpretation, and writing-original draft. All authors contributed to the systematic review of the published findings and sources. They all read and approved the final manuscript.

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Correspondence to Wenting Qi, Meng Xu or Jianxiong Li.

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All methods were performed in accordance with the relevant guidelines and regulations. The protocol for this study was approved by the Ethics Committee of the Fourth Medical Center of Chinese PLA General Hospital (agreement number: 2024KY0112-KS001).

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Liu, Y., Han, Y., Guo, Z. et al. Unveiling the role of the extracellular matrix in the osteosarcoma tumor microenvironment through integrated transcriptomics and experimental validation. Cancer Gene Ther (2025). https://doi.org/10.1038/s41417-025-00970-0

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