Protocol for generating a co-culture of macrophages with breast cancer tumoroids

Cancer progression and treatment outcomes are heavily influenced by the tumor microenvironment (TME), especially through immune cell interactions. Here, we present a protocol for generating co-cultures of tumoroids with macrophages, either semi-liquid or Matrigel-embedded. We describe steps for macrophage preparation, co-culture establishment, and medium adjustments to support cell viability and function. While optimized for breast cancer models, this protocol can be adapted to other tumor types with appropriate medium adjustments. For complete details on the use and execution of this protocol, please refer to Raffo-Romero et al. 1 • Steps for three co-culture models of breast cancer tumoroids and macrophages • Details for isolating and detaching primary macrophages • Guidance on culturing breast cancer tumoroids from biopsy samples • Co-culture influences drug response, emphasizing the necessity for model complexity Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Cancer progression and treatment outcomes are heavily influenced by the tumor microenvironment (TME), especially through immune cell interactions. Here, we present a protocol for generating co-cultures of tumoroids with macrophages, either semi-liquid or Matrigel-embedded. We describe steps for macrophage preparation, co-culture establishment, and medium adjustments to support cell viability and function. While optimized for breast cancer models, this protocol can be adapted to other tumor types with appropriate medium adjustments.