Protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments.

3D culture models better replicate tumor structure and interactions than 2D methods, enhancing cancer therapy research. Here, we present a protocol to develop a 3D mouse autologous lung tumor-immune spheroid co-culture model to advance radiotherapy-based treatments. We describe steps for optimizing tumor spheroids, isolating CD4+ T cells and monocytes, and differentiating T regulatory (Treg) cells and M2-like macrophages. We then detail procedures for seeding tumor-immune spheroids, performing fluorescence-activated cell sorting (FACS) analysis, and characterizing spheroids, including their response to radiotherapy.