Work

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

Year: 2025

Type: article

Abstract: 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. • Optimization of a 3D culture technique based on TSp formation in agarose molds • Enrichment of TSp with T regulatory cells and M2-like macrophages • Characterization of TImS using cell trackers and flow cytometry • Assessment of radiotherapy efficacy in TImS in a unique autologous lung cancer model Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2025.104021