Assessing cancer cell heterogeneity in patient-derived tumoroids

Here we detail a method for the exploration of patient-derived xenografts (PDX) based on organoids (PDX-O), using antibodies validated for immunohistochemistry. Importantly this approach can be used to characterize the response of malignant cells to antineoplastic treatments. Cancer progression and metastasis reflect tumor heterogeneity and plasticity. Here we analyzed cells in PDX grown as spheroids or tumoroids embedded in collagen/basement membrane extract (BME) matrix. The use of 3D cellular models instead of 2D allows the inclusion in the study of non-adherent tumors. Even if the cells are adherent the spheroid and tumoroids conformation or organization in space and cellular polarity differ for 2D. This difference can change the response to treatment. Adding a matrix component is an important addition to the tumoral microenvironment that can change also the cellular response to treatment. We combined this tridimensional model with a multiplexed staining protocol to analyze tumor cell differentiation and responses to drugs. We analyzed paraffin embedded organoid sections, allowing to analyze the core of cell aggregates, and analyzed cell plasticity, differentiation and proliferation with and without treatment with palbociclib. Spheroids included an outer layer of luminal cytokeratin (CK) 8-positive cells surrounding a mixed population of cells expressing both CK5 and CK8. Palbociclib treatment resulted in a proliferation arrest, linked to dedifferentiation of the cancer cells that switched to a mostly basal phenotype, supporting a hybrid CK5/CK8 phenotype. In summary, this method offers a simple and versatile strategy to analyze tumor cell responses to drugs, using routine antibodies validated for immunohistochemistry and multiplex analysis.