Subpopulations of organoid-forming cells have different motility

Cancer stem cells from oropharyngeal squamous cell carcinoma (OPSCC) have the ability to self-renew and differentiate into heterogeneous three-dimensional structures carrying features of tumor cells. Here, we describe a simple and label-free method for generating tumor organoids, and imaging them using live digital holographic microscopy (DHM) on the basis of the phase shift caused by light passing through the cells. We show early events of cell aggregation during tumor-organoid formation, and display their heterogeneity in terms of optical parameters up to an optical volume of 10⁵ μm³. Lastly, by sorting OPSCC epithelial cells, we demonstrate that CD44⁺ cells displayed greater motility and tumor-forming capacity than those of CD44^- cells. These results were in line with previous reports highlighting increased invasive and tumorigenic potential in tumor cells expressing high levels of CD44. Our method provides insight into the formation of tumor organoids, and could be used to assess stemness-associated biomarkers and drug screenings on the basis of tumor organoids.