Immunosuppressive tumor microenvironment of osteosarcoma

Background - Osteosarcoma (OS) is the most common malignant bone tumor in children. OS is characterized by a high degree of genomic instability, resulting in copy-number alterations and genomic rearrangements with no disease-defining recurrent mutations. Given the diverse genomic landscape of OS and the difficulty of identifying druggable therapeutic targets, use of immunotherapy techniques appears lucrative. However, clinical trials based on molecular characterization have failed to find new effective therapies, and outcomes have not improved over the last 40 years. Materials/Methods - We performed single-cell RNA sequencing (scRNA-seq) using the 10x Genomics Chromium platform on six fresh tumor biopsy samples from pediatric OS patients. Raw data was processed using 10x CellRanger to produce transcript read counts for each cell. After filtering low-quality cells and doublet removal, counts were normalized using Seurat, and cells were integrated across samples with Harmony. Data was combined with a previously-published OS scRNA-seq cohort of six samples (GSE162454). Two additional OS samples were profiled using 10x Genomics Visium spatial transcriptomics for validation of discovered subtypes and to add spatial context. Results - Clustering identified 16 major cell types based on expression of canonical cell markers. Several immunosuppressive cell types were identified via subclustering of major cell types, including neutrophil myeloid-derived suppressor cells (MDSCs), regulatory and exhausted T-cells, and LAMP3+ dendritic cells. Markers for the cell types found in OS were identified for further validation using imaging techniques, including Visium spatial transcriptomics. We performed deconvolution using the scRNA-seq cell identities to examine colocalization of discovered cell types. Overall, the discovered clusters were common between patients, showing consistent cell type proportions. However, we found patient-specific differences in the frequency of some cell types, with one sample showing a higher proportion of T-cells along with increased presence of colocalized IFN-stimulated macrophages, and the other with a greater presence of neutrophils/MDSCs. Conclusions - Using single-cell transcriptomics, we were able to discover the presence of multiple immunosuppressive cell subtypes of neutrophils, T-cells, and dendritic cells. Additionally, spatial transcriptomics revealed multiple similar clusters between samples, and common colocalization of the discovered cell types within those clusters. However, differences in T-cell presence and interferon induction may be indicative of patient-specific immunogenicity in osteosarcoma tumors. ### Competing Interest Statement The authors have declared no competing interest.