Protocol for sterile tumor cell implantation into the spleen or cecal wall of germ-free mice

Establishing tumor models in germ-free (GF) mice is essential to study the effects of microbiome on cancer progression and treatment responses. Here, we present a protocol for the intrasplenic and orthotopic cecal wall implantation of colorectal cancer cells in GF mice under sterile conditions. Intrasplenic injection models late-stages of liver metastasis, whereas orthotopic cecal wall implantation recapitulates the complete metastatic cascade. We describe procedures for material preparation, sterile surgery, and sterility monitoring to ensure successful tumor establishment while maintaining GF status.