(A) Workflow of preparation of TCS-PCLS by implementation of A549-positive GFP on nontumor/healthy PCLS. Created in Biorender.com. (B) Healthy PCLS was seeded with A549-GFP cells. After 24 hours, tissue was fixed and imaged with the Leica Thunder Imager using the lung autofluorescence (red), and the GFP signal emitted from A549 cells (green). Scale bar: 500 μm (right), 100 μm (left, magnification). (C) Healthy PCLS was cultured with A549-GFP spheroids for 24 hours. Spheroid outgrowth was imaged with the Leica SP8 confocal microscope using the Lung autofluorescence (red) and the GFP signal (green). Scale bar: 250 μm. (D) Healthy PCLS was seeded with A549-GFP cells. Time lapse imaging was done with the Keyence New All-in-One Fluorescence Microscope BZ-X800 in a stage top incubator for 24 hours. Scale bar: 500μm. (E) Healthy PCLS was seeded with A549-GFP cells and cultured for 24 hours. Prior fixation, 20 μM EdU was added overnight and visualized using the Click-iT EdU Kit. Slices were imaged using the Leica SP8 confocal microscope for the Lung autofluorescence (red), GFP signal (green), and EdU incorporation (magenta). Scale bar: 100 μm. (F) Healthy PCLS was seeded with A549-GFP and then cultured in different types of macrophage-conditioned medium (CM) for 24 hours, including naive unstimulated M0 macrophages, tumor-promoting M2 macrophages (stimulated with IL4 for 24 hours), antitumor M1 macrophages (stimulated with LPS and IFN-γ for 24 hours). Slices were imaged using the Leica SP8 confocal microscope. Scale bar: 100 μm.