Hypoxia is a common feature present in solid tumors, which promotes the development of invasive and aggressive malignant cells that resist both conventional cancer treatments and immunotherapy. Standard cell culture techniques do not adequately replicate this complex development of solid tumors. Our purpose was to investigate the mechanisms that limit the effectiveness of CAR T therapy in solid tumors, particularly osteosarcoma (OS). In this study, we present the results using NKG2D-CAR T against OS cell lines in a hypoxic 3D microenvironment.

To mimic physiological environment, we facilitate cell-cell OS interactions in ultra-low adherent plate, which facilitate spheroid formation. OS spheroids and NKG2D-CAR T cells were co-culture in hypoxic environment to test the cytotoxicity, cytokines expression and T cells activation patterns. Immune checkpoints markers and NKG2D ligands were also analyses under hypoxia in OS cells, which can modulate the efficacy of NKG2D-CAR T therapy

We demonstrated that NKG2D-CAR T cells were effective in hypoxic spheroids, expressing cytokines such as INFg and TNFa. NKG2D CAR T cells activation and inhibitory pathways such as CD69, CD25, PD1 and TIM3 were similar expressed in hypoxic environment and immune checkpoints inhibitory pathways were maintained in hypoxia culture, as well as NKG2D ligands

Overall, hypoxic OS spheroids represent a valuable tool to study the CAR T tumour and anticipate possible clinical outcomes for patients.