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.