Fig. 4

CAR-modified macrophages effectively eradicate tumor patient-derived organoids (PDOs). Cultivation of gallbladder cancer PDOs (A) and pancreatic cancer PDOs (B). The morphology of the PDOs was examined via bright-field microscopy. Additionally, hematoxylin and eosin (HE) staining and immunohistochemical (IHC) staining were performed to assess the expression of HER2 and CA199 in the PDOs. Scale bars represent 20 μm for bright-field and HE-stained images and 50 μm for IHC-stained images. C Confocal imaging of the accumulation of CAR-modified macrophages around PDOs. PDOs were cocultured with CAR- and CAR-shSIRPα-treated macrophages (green) for 12 h. Nuclei were stained prior to confocal imaging. Scale bars represent 20 μm. D Phagocytosis of PDOs by CAR-shSIRPα macrophages. PDOs were stained with CellTracker (red) and cocultured with CAR-shSIRPα macrophages (green) for 48 h before confocal imaging. Scale bars represent 50 μm. E Cytotoxic effects of CAR-shSIRPα macrophages on PDO cells. PDOs labeled with CellTracker (red) were cocultured with CAR-shSIRPα macrophages that did not express GFP for 48 h, after which Apopxin™ Green was added to identify apoptotic cells before confocal imaging. Scale bars represent 50 μm. Destruction of the PDO structure by CAR-modified macrophages. Gallbladder cancer PDOs (F, scale bars represent 100 μm) or pancreatic cancer PDOs (G, scale bars represent 20 μm) were cocultured with GFP, CAR, and CAR-shSIRPα macrophages. Bright-field microscopy and HE were performed at the specified time points. H–K Identification of proinflammatory cytokines in the supernatants of the cocultures. Gallbladder cancer PDOs (H, J) and pancreatic cancer PDOs (I, K) were cocultured with GFP, CAR, and CAR-shSIRPα macrophages for 72 h. The supernatant was analyzed via ELISA to measure the levels of IL-1β (H, I) and TNF-α (J, K). For all panels, *P < 0.05, **P < 0.01, ***P < 0.001