1:Instituto de Salud Carlos III

Standard two-dimensional cell culture techniques do not reproduce the dynamic and complex progression of osteosarcoma (OS) and fail to mimic cell-to-cell or cell-to-microenvironment interactions. Current three-dimensional (3D) models, also called organoids or spheroids, provide powerful models for studying cancer treatment, including immunotherapies. Despite the efficacy of chimeric antigen receptor (CAR) T cells in haematological tumours, sarcoma patients remain resistant to this immunotherapy. To overcome these limitations, various 3D cell culture techniques have been developed. Here, we describe an approach to study NKG2D-CAR T cells against OS cell lines, such as 143B or MnnG-hOS. We facilitated cell-cell interactions in scaffold-free wells with U-shaped bottoms. NKG2D ligands, tumour cytotoxicity, cytokines release and phenotype of CAR T cells were analysed in the organoid model. We showed that 3D cultures decreased NKG2D ligands, which are targets of our NKG2D-CAR T cells. Despite this ligand depletion, we demonstrate that NKG2D-CAR T cells were able to infiltrate OS organoids and induce anti-tumour activity. Infiltrating NKG2D-CAR T cells have different activation phenotype compared to non-infiltrating cells. In this regard, differences in cytokine expression were also observed in 3D co-cultures. In conclusion, OS organoids represent a valuable tool to study CAR T-tumour interactions because they represent a physiologically relevant model.