Multiplex Imaging Mass Cytometry Reveals Prognostic Immunosuppressive Subpopulations and Macrophage-Driven Metastasis in Osteosarcoma

<b>Background:</b> 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). <b>Methods:</b> 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. <b>Results:</b> 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, <i>p</i> = 0.0244) and recurrence-free survival (RFS, <i>p</i> = 0.0040). Notably, closer spatial proximity between M2 macrophages and immunosuppressive cells (<i>p</i> = 0.0248) or Ki-67⁺ cells (<i>p</i> = 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 (<i>p</i> = 0.0140). Luminex analysis identified M2-derived MIP-1α (CCL3) as a candidate chemokine driving OS cell metastatic potential. <b>Conclusions:</b> 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.