Background:Metastasis continues to be a leading cause of mortality in osteosarcoma (OS) among pediatric and young adult populations, with few effective therapeutic options available. Despite immunotherapy advancements, its efficacy in OS is hindered by an incomplete understanding of the immunosuppressive tumor microenvironment (TME).Methods:We utilized multiplex imaging mass cytometry and phenoplexing to characterize immune and stromal cell populations within the TME of a tissue microarray comprising 51 primary OS tumors. The prognostic significance of TME cell abundance and spatial cell–cell distance was evaluated using Kaplan–Meier and Cox regression analyses. To investigate macrophage functionality in vivo, we employed orthotopic xenograft mouse models by co-injecting THP-1-derived M0 or M2 macrophages with 143B OS cells to assess their impact on tumor growth and pulmonary metastasis. Mechanisms of macrophage-mediated metastasis were explored using Luminex, ELISA, and transwell migration assays.Results:Our results showed that macrophages dominated the TME, with M0 and M2 subtypes significantly outnumbering M1 macrophages (M1) and other myeloid cells. T cells and myeloid-derived suppressor cells (MDSC) were the second and third most abundant immune populations, respectively. Among stromal cells, endothelial cells predominated over fibroblasts. While individual immunosuppressive cell populations (M2, MDSC, and Treg) showed no direct correlation with clinical outcomes, the collective abundance of M2 and MDSC was significantly associated with reduced metastasis-free survival (MFS,p= 0.0244) and recurrence-free survival (RFS,p= 0.0040). Notably, closer spatial proximity between M2 macrophages and immunosuppressive cells (p= 0.0248) or Ki-67+cells (p= 0.0321) correlated with decreased MFS, suggesting the formation of an M2-centric immunosuppressive and pro-tumor hub. In vivo, co-injection of M2 macrophages with 143B cells significantly enhanced pulmonary metastasis (p= 0.0140). Luminex analysis identified M2-derived MIP-1α (CCL3) as a candidate chemokine driving OS cell metastatic potential.Conclusions:This study provides a high-resolution map of the OS TME, highlighting the prognostic significance of M2 and immunosuppressive cell interactions in driving metastasis, potentially through MIP-1α signaling. These findings establish a foundation for developing targeted immunotherapies to improve outcomes in metastatic OS.
背景：转移仍是儿童及青少年骨肉瘤（OS）患者的主要致死原因，且目前缺乏有效治疗手段。尽管免疫治疗取得进展，但由于对免疫抑制性肿瘤微环境（TME）的认识不足，其在骨肉瘤中的疗效受到限制。 方法：我们采用多重成像质谱流式技术与表型组学分析，对包含51例原发性骨肉瘤组织的组织芯片进行TME内免疫细胞及基质细胞群体的特征解析。通过Kaplan-Meier生存分析与Cox回归模型评估TME细胞丰度及细胞间空间距离的预后意义。为探究巨噬细胞在体内的功能，我们建立原位移植瘤小鼠模型，将THP-1来源的M0或M2型巨噬细胞与143B骨肉瘤细胞共注射，评估其对肿瘤生长及肺转移的影响。采用Luminex多重检测、ELISA及Transwell迁移实验探索巨噬细胞介导转移的机制。 结果：研究显示巨噬细胞在TME中占主导地位，其中M0与M2亚型数量显著超过M1型巨噬细胞及其他髓系细胞。T细胞与髓源性抑制细胞（MDSC）分别为第二和第三丰富的免疫群体。基质细胞中内皮细胞数量多于成纤维细胞。虽然单个免疫抑制细胞群体（M2、MDSC、Treg）与临床结局无直接相关性，但M2与MDSC的联合丰度与无转移生存期（MFS，p=0.0244）及无复发生存期（RFS，p=0.0040）缩短显著相关。值得注意的是，M2巨噬细胞与免疫抑制细胞（p=0.0248）或Ki-67阳性细胞（p=0.0321）的空间距离越近，MFS越短，提示形成了以M2为核心的免疫抑制促瘤枢纽。体内实验证实，M2巨噬细胞与143B细胞共注射可显著促进肺转移（p=0.0140）。Luminex分析鉴定出M2来源的MIP-1α（CCL3）是驱动骨肉瘤细胞转移潜力的关键趋化因子。 结论：本研究绘制了骨肉瘤TME的高分辨率图谱，揭示了M2与免疫抑制细胞相互作用通过MIP-1α信号通路驱动转移的预后价值，为开发靶向免疫疗法以改善转移性骨肉瘤预后奠定了理论基础。
肿瘤(癌症)患者之家