Identification of canine melanoma cancer stem cells

Epithelial-mesenchymal transition (EMT) is a process, where epithelial cells lose their typical properties but gain mesenchymal characteristics. Additionally, EMT is associated with a process that promotes the transition of epithelial tumour cells to a cancer stem cell (CSC) like phenotype. CSCs share typical stem cell properties such as self-renewal and multidirectional differentiation. Furthermore, they are resistant to stress factors such as DNA damage or chemo- and radiation therapy. Canine oral malignant melanoma (cOMM) has a high metastatic potential and is a common malignancy in dogs. In cOMM, only scarce information is available on the presence and pathogenic role of CSCs. Therefore, we assessed cOMM cells for expression of two different clusters of differentiation, i.e., CD44 and CD271, that are confirmed markers for human CSCs. CD44 is a very abundant marker for CSCs in general, and CD271 is one that relates to metastases, especially in the brain. It is currently assumed that CSCs are CD271-positive cells within the CD44-positive cell compartment, which are hyperproliferating and show higher resistance to treatment than CD44-positive CD271-negative cells. To identify CSCs in cOMM, a total of nine primary cOMM cell lines that have been previously established from patient-derived primary cOMM and cOMM metastases were cultured in 2D and 3D (spheres). COMM cells and spheres were analysed by immunocyto- and immunohistochemical staining and flow cytometry using antibodies specifically binding to CD44 and CD271. CD44-positive cells were detected in all nine cOMM cell lines. CD271 was expressed in three 2D- and two 3D cultured cell lines, of which only one, cRGO1, contained a CD44-positive CD271-positive subpopulation in both cultivation systems. This suggests that during EMT potential CSC properties can change depending on which influences the cells are exposed to.