Serial Orthotopic Transplantation of Epithelial Tumors in Single-Cell Suspension

Orthotopic transplantation of tumor tissue into recipient mice has long been established to study the role of the microenvironment in tumorigenesis and metastasis. Many of these transplantation assays involve the surgical implantation of an undissociated piece of tumor tissue. However, dissociation of tumor tissue into a single cell suspension prior to orthotopic transplantation enables the injection of fewer cell numbers, the selection of tumor-initiating populations by specific purification using antibody staining and fluorescence-activated cell sorting, and the analysis of tumor-forming efficiency. In this chapter, we provide a method to perform serial transplantation of tumor cells into their niche of origin. Visualization of the location of transplanted tumor cells is essential to confirm the success of the transplant as well as the viability of transplanted cells. We also describe an optimized immunofluorescence protocol to visualize tumor cells shortly after transplantation. This serial transplantation protocol allows for an experimental tumorigenesis assay to more closely mimic spontaneous tumor formation and is applicable to many microenvironments.