3D Tumor-Macrophage Co-Culture Spheroids

Three-dimensional (3D) co-cultured multicellular tumor spheroids of tumor cells (4T1 cell line) and macrophages (RAW264.7 cell line) were developed using a liquid-overlay system according to the culture scheme illustrated in Figure 6A.30 (link) RAW264.7 cells were stained with 10 μg/mL 1,1‘-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI) prior to spheroid formation. 4T1 cells (1×10⁴ cells) and DiI-labeled RAW264.7 cells (0.5×10⁴ cells) were mixed with DMEM containing 10% FBS and 2.5% Matrigel, seeded in a 96-well round bottom plate (Corning, Corning, NY, USA), and centrifuged at 1000× g for 10 min. After 3 days, separate groups of co-cultured tumor spheroids were incubated with Lipo, PEG-Lipo, and Man-PEG-Lipo for 8 h. At the end of incubation, the co-cultured tumor spheroids were washed three times with cold PBS, and fixed with 4% PFA for 1 h at room temperature. The fluorescence intensity of different depths of the co-cultured tumor spheroids was determined using CLSM.