Protocol for analyzing immune cell infiltration in 3D pancreatic heterotypic spheroids composed of tumor cells and cancer-associated fibroblasts

We present a protocol to generate and analyze 3D cancer models composed of tumor cells, cancer-associated fibroblasts (CAFs), and immune cells that mimic the complexity of the pancreatic ductal adenocarcinoma (PDAC) stroma. The protocol includes steps and conditions for establishing 3D co-cultures, embedding for histological analysis, and flow cytometry analysis for the immunophenotyping and characterization of tumor-infiltrating immune cells. These models are valuable for testing strategies and studying the roles of CAFs and immune cells in tumor growth, chemoresistance, and immunosuppression.