Protocol for generation of a 3D humanized lung-tumor-on-a-chip model of immune-hot and immune-cold microenvironments

The tumor microenvironment (TME) modulates immune responses and clinical outcomes. Here, we present a protocol to establish a fully humanized immune-hot and -cold lung TME using the Emulate organ-on-a-chip system by culturing human lung microvascular endothelial cells and human lung tumor cells. We describe steps for perfusing human peripheral blood mononuclear cells (PBMCs), visualizing immune cell recruitment, and analyzing immune infiltration across TME phenotypes through immunofluorescence, flow cytometry, or secretome analysis. This protocol can be adapted to model the TME in different cancer types.