Immunosuppressive tumor microenvironment of osteosarcoma [Spatial transcriptomics]

Osteosarcoma is the most common malignant bone tumor in children, characterized by a high degree of genomic instability, resulting in copy-number alterations and genomic rearrangements without disease-defining recurrent mutations. Clinical trials based on molecular characterization have failed to find new effective therapies or improve outcomes over the last 40 years. To better understand the immune microenvironment of osteosarcoma, we performed single-cell RNA sequencing on six tumor biopsy samples, combined with a previously-published cohort of six samples. Additional osteosarcoma samples were profiled using spatial transcriptomics for validation of discovered subtypes and to add spatial context. Analysis revealed immunosuppressive cells, including myeloid-derived suppressor cells (MDSCs), regulatory and exhausted T-cells, and LAMP3+ dendritic cells. Using cell-cell communication modeling, we identified robust interactions between MDSCs and other cells, leading to NF-?B upregulation and an immunosuppressive microenvironment, as well as interactions involving regulatory T-cells and osteosarcoma cells that promoted tumor progression and a proangiogenic niche. Overall design: Formalin-fixed, paraffin-embedded (FFPE) samples of two primary OS pre-treatment biopsy tissue samples (Patients A and B) were obtained from CCMC and stored at 4?°C in the dark. Prior to Visium transcriptomics, RNA quality of FFPE samples was determined by DV200 score using Agilent (Santa Clara, California) TapeStation 4200 High Sensitivity DNA ScreenTape. Tissue blocks with DV200 scores above 50% were used for downstream processing. Briefly, FFPE sections were placed on a 10x Visium FFPE Gene Expression slide, deparaffinized, H&E stained, then imaged in brightfield using a NanoZoomer SQ (Hamamatsu Photonics, Shizuoka, Japan) slide scanner, followed by incubation with human-specific probe sets provided by the manufacturer for subsequent mRNA labeling and library generation per the manufacturer's protocol (10x Genomics, CG000407). Libraries were pooled for sequencing on an Illumina NovaSeq 6000 200-cycle S4 flow cell using a 28-10-10-90 read configuration, targeting 100,000 read pairs per spot covered by tissue.