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.
Authors
- Flávia Castro (ORCID: https://orcid.org/0000-0002-2273-2806)
- Alfonso Calvo (ORCID: https://orcid.org/0000-0003-4074-4242)
- Javier Aristu (ORCID: https://orcid.org/0000-0002-0382-2363)
- María José Oliveira (ORCID: https://orcid.org/0000-0002-0724-0272)
- Sofía Torres Quintas (ORCID: https://orcid.org/0000-0002-4190-3527)
- Miriam Redrado (ORCID: https://orcid.org/0000-0003-0120-0779)
- Diego Serrano (ORCID: https://orcid.org/0000-0002-3729-9195)
- Nerea Otegui (ORCID: https://orcid.org/0009-0009-7470-0847)
- Sergio León (ORCID: https://orcid.org/0009-0004-9941-5069)
Institutions
- Universidade do Porto (PT)
- Clinica Universidad de Navarra (ES)
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto (PT)
- Universidad de Navarra (ES)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-08-12
- DOI
- https://doi.org/10.1016/j.xpro.2025.104021
- Primary Topic
- Cancer Cells and Metastasis
- Type
- article
- Field-Weighted Citation Impact
- 0.00