In vitro models to mimic tumor endothelial cell-mediated immune cell reprogramming in lung adenocarcinoma.

Tumor endothelial cells (TECs) play a critical role in regulating immune responses within the tumor microenvironment (TME). However, the mechanisms by which TECs modulate immune cell population remain unclear, particularly in non-small cell lung cancer (NSCLC). Here, we investigated how NSCLC cells tweak normal endothelial cells (NECs) into TECs and the subsequent effects on immune regulation. NECs were cocultured with various NSCLC cell lines, using 2D and 3D coculture models to evaluate TEC-mediated effects on immune cells. We show that direct coculture led to significant transcriptomic, proteomic and kinomic alterations in TECs, especially in pro-inflammatory pathways. We identified a downregulation of the co-stimulatory molecule OX40L in TECs compared to NECs, suggesting impaired T-cell proliferation support. While TECs showed a limited effect on CD8(+) T-cell activation, TECs supported CD4(+) T-cells polarization into Treg and Th22 subsets. Moreover, TECs also promoted M2-like macrophages polarization, thereby potentially contributing to the TME immunosuppression. State-of-the-art single-cell RNA sequencing of 3D multicellular tumor spheroids (MCTS) revealed formidable heterogeneity in the tumor cells and cancer-associated fibroblast compartments. It also unveiled distinct TEC subpopulations, including an inflammatory subset with an unfolded-protein response signature. This TEC cluster was absent in 2D-cultured NECs but present in freshly isolated and 2D-cultured TECs from NSCLC patients. Importantly, we identified a perivascular M2-like macrophage subset within MCTS that is in close contact with TECs, and is predicted to interact with them through MIF signaling. In conclusion, TECs in NSCLC tumors play a pivotal role in remodeling the TME immune landscape by promoting immune suppression. This study highlights the complex immunoregulatory functions of TECs within different in vitro models that mimic aspects of the TME. Our data may provide new insights into potential therapeutic strategies targeting TECs or regulatory signaling to improve the efficacy of immunotherapy in NSCLC.