Rationale: Osteosarcoma (OS) is the most common primary bone tumor with a poor prognosis, but the detailed mechanism is still unclear. A comprehensive investigation of tumor microenvironment (TME) of OS might help find effective anti-tumor strategies. Single-cell transcriptomics is a powerful new tool to explore TME. Therefore, this study is designed to investigate the TME and gene expression pattern of primary and recurrent OS at the single-cell level. Methods: The single-cell RNA sequencing and bioinformatic analysis were conducted to investigate the cellular constitution of primary, recurrent, and lung metastatic OS lesions according to the datasets of GSE152048 and GSE162454. TIMER database was used to investigate the role of LOX in the prognosis of sarcoma. The functions of related cells and markers were further confirmed by in vitro and in vivo experiments. Results: Cancer associated fibroblasts (CAFs) were found with a higher infiltrating level in recurrent OS, and were enriched in the epithelial-mesenchymal transition (EMT) pathway. CAFs showed remarkably increased expression of LOX, which might lead to EMT and poor prognosis of OS. Mechanically, LOX regulated the function of CAFs and macrophage polarization to remodel the tumor immune microenvironment. Moreover, LOX inhibitor could inhibit migration and promote apoptosis of OS both in vitro and in vivo. Conclusions: This study revealed the heterogeneity of recurrent OS and highlighted an innovative mechanism that CAFs regulate EMT of OS via LOX. Targeting LOX of CAFs showed promising efficacy in remodeling TME and treating recurrent OS.Keywords: Cancer associated fibroblasts; Osteosarcoma; Single-cell transcriptomics; Tumor immune microenvironment.

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乐备实（上海优宁维生物科技股份有限公司旗下全资子公司），是国内专注于提供高质量蛋白检测以及组学分析服务的实验服务专家，自2018年成立以来，乐备实不断寻求突破，公司的服务技术平台已扩展到单细胞测序、空间多组学、流式检测、超敏电化学发光、Luminex多因子检测、抗体芯片、PCR Array、ELISA、Elispot、PLA蛋白互作、多色免疫组化、DSP空间多组学等30多个，建立起了一套涵盖基因、蛋白、细胞以及组织水平实验的完整检测体系。

 
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